Secretion of carrier-bound proteins into the periplasma and the extracellular space

ABSTRACT

The present invention relates to processes for producing S-layer proteins and modified S-layer proteins in Gram-negative host cells.

This Application is a 371 of PCT/EP98/04723 filed on Jul. 27, 1998,which claims benefit of German Application 197 32 829.6 filed on Jul.30, 1997.

The present invention relates to processes for producing carrier-boundproteins, in particular S-layer proteins and modified S-layer proteinsin pro- or eukaryotic host cells.

Crystalline bacterial cell surface layers (S-layers) form in manyeubacteria and in most archaebacteria of the outermost cell wallcomponent (Sleytr et al. (1988), Crystalline Bacterial Cell SurfaceLayers, Springer Verlag Berlin; Messner and Sleytr. Adv. Mikrob.Physiol. 33 (1992), 213-275). Most of the S-layer proteins known atpresent are composed of identical proteins or glycoproteins which haveapparent molecular weights in the range from 40,000 to 220,000. Thecomponents of S-layers are self-assembling and most lattices haveoblique (p2), square (p4) or hexagonal (p6) symmetry. The functions ofbacterial S-layers are still not completely known but, on the basis oftheir localization on the cell surface, it is likely that the porouscrystalline S-layers act mainly as protective coverings, molecularsieves or for promoting cell adhesion and surface recognition.

Genetic data and sequence information are known for various S-layergenes from microorganisms. A review is to be found in Peyret et al.,Mol. Microbiol. 9 (1993), 97-109. Express reference is made to thesedata. The sequence of the gene sbsA coding for the S-layer protein ofB.stearothermophilus PV72 and a method for cloning it are indicated byKuen et al. (Gene 145 (1994), 115-20). B.stearothermophilus PV72 is aGram-positive bacterium which is covered by a hexagonally arrangedS-layer. The main component of the S-layer is a 128 kd protein which isthe commonest protein in the cell, comprising approximately 15% of thetotal protein constituents. Various strains of B.stearothermophilus havebeen characterized and differ in the type of, S-layer lattice, themolecular weight and the glycosylation of the S-layer components(Messner and Sleytr (1992), supra).

German Patent Application DE-A 44 25 527 discloses the signalpeptide-encoding section of the S-layer gene of B.stearothermophilus andthe amino acid sequence derived therefrom. The cleavage site between thesignal peptide and the mature protein is located between position 30 and31 of the amino acid sequence. The signal peptide-encoding nucleic acidcan be operatively linked to a protein-encoding nucleic acid and usedfor the recombinant production of proteins in a process in which atransformed host cell is prepared, the host cell is cultivated underconditions which lead to expression of the nucleic acid and toproduction and secretion of the polypeptide encoded thereby, and theresulting polypeptide is isolated from the culture medium. The hostcells mentioned as preferred are prokaryotic organisms, in particularGram-positive organisms of the genus Bacillus.

The international Patent Application PCT/EP97/00432 proposes therecombinant production of S-layer proteins and modified S-layer proteinsin the cytoplasm of Gram-negative host cells.

It has now been found, surprisingly, not only that the recombinantproduction of S-layer proteins is possible in the cytoplasm ofGram-negative prokaryotic host cells, but also that recombinantexpression comprising integration in the outer or the cytoplasmicmembrane, secretion into the periplasm or/and secretion into theextracellular space can be carried out. It has additionally been foundthat recombinant expression of S-layer proteins also takes place in theeukaryotic host cells.

A first aspect of the present invention is thus a process for producingS-layer proteins, which comprises

(a) preparing a Gram-negative prokaryotic host cell which is transformedwith a nucleic acid which codes for an S-layer protein and isoperatively linked to a signal sequence which codes for a peptide whichbrings about integration of the S-layer protein in the outer membrane ofthe host cell, integration of the S-layer protein in the cytoplasmicmembrane of the host cell, secretion of the S-layer protein into theperiplasmic space of the host cell or/and secretion into the mediumsurrounding the host cell,

(b) cultivating the host cell under conditions leading to expression ofthe nucleic acid and to production of the polypeptide encoded thereby,and

(c) where appropriate isolating the resulting polypeptide from the outermembrane of the host cell, from the cytoplasmic membrane of the hostcell from the periplasmic space of the host cell or/and from the mediumsurrounding the host cell.

A second aspect of the present invention is a process for producingS-layer proteins, which comprises

(a) preparing a eukaryotic host cell which is transformed with a nucleicacid which codes for an S-layer protein and is preferably operativelylinked to a signal sequence which brings about integration of theS-layer protein in the cytoplasmic membrane of the host cell,integration of the S-layer protein into an organelle of the host cellor/and secretion into the medium surrounding the host cell,

(b) cultivating the host cell under conditions leading to expression ofthe nucleic acid and to production of the polypeptide encoded thereby,and

(c) where appropriate isolating the resulting polypeptide from thecytoplasmic membrane of the host cell, from an organelle of the hostcell or/and from the medium surrounding the host cell.

It has been found, surprisingly, that secretion of any heterologousS-layer proteins, including recombinant S-layer proteins, into theperiplasmic space of a Gram-negative host cell or even secretion intothe medium surrounding the host cell is possible. This entails theS-layer protein being formed in the periplasm of the host cell not inthe form of unordered inclusion bodies but, unexpectedly, in the form ofordered monomolecular layers. In addition, anchoring of heterologousS-layer proteins in the outer or the cytoplasmic membrane ofGram-negative host cells is possible.

S-layer proteins can also be expressed in functional form in eukaryoticcells such as, for example, mammalian cells or yeast. Glycosylationtakes place in the case of recombinant S-layer proteins having aeukaryotic fusion portion. In addition, glcosylation may take place inthe S-layer protein portion itself.

The process according to the invention makes it possible preferably toexpress S-layer genes derived from B.stearothermophilus PV72,inparticular to express the S-layer genes sbsA and sbsB. In addition,however, it is also possible to express S-layer genes from otherorganisms (cf., for example, Peyret et al., (1993) supra) by the processaccording to the invention.

The nucleotide sequence of the gene coding for the mature SbsA proteinis indicated in SEQ ID NO. 1 from position 91-3684. The relevant aminoacid sequence is depicted in SEQ ID NO. 2. The nucleotide sequence forthe gene coding for the mature SbsB protein is indicated in SEQ ID NO. 5from position 94-2763. The a relevant amino acid sequence is depicted inSEQ ID NO. 6.

In a first preferred embodiment (sbsA), the nucleic acid coding for anS-layer protein is selected from

(i) a nucleic acid which comprises the nucleotide sequence shown in SEQID NO. 1 from position 91 to 3684,

(ii) a nucleic acid which comprises a nucleotide sequence correspondingto the nucleic acid from (i) within the scope of the degeneracy of thegenetic code, and

(iii) a nucleic acid which comprises a nucleotide sequence hybridizingwith the nucleic acids from (i) or/and (ii) under stringent conditions.

In a second preferred embodiment (sbsB), the nucleic acid coding for anS-layer protein is selected from

(i) a nucleic acid which comprises the nucleotide sequence shown in SEQID NO. 5 from position 94 to 2763,

(ii) a nucleic acid which comprises a nucleotide sequence correspondingto the nucleic acid from (i) within the scope of the degeneracy of thegenetic code, and

(iii) a nucleic acid which comprises a nucleotide sequence hybridizingwith the nucleic acids from (i) or/and (ii) under stringent conditions.

The term “stringent hybridization” means for the purpose of the presentinvention that hybridization still occurs even after washing at 55° C.,preferably 60° C., in an aqueous low-salt buffer (for example 0.2×SSC)(see also Sambrook et al. (1989), Molecular Cloning. A LaboratoryManual.

Gram-negative prokaryotic host cells are used in the first aspect of theinvention. In this case, surprisingly, an S-layer protein assembled inan ordered structure is obtained in the periplasm. The host cellspreferably used are enterobacteria, in particular E.coli. Examples ofsuitable E.coli strains are DH5α (sup E44, Δ lac U169, hsdR17, recA1,endA1, gyr A96, thi-1, rel A1; Hanahan, J. Mol. Biol. 166 (1983),557-580) and HB 2151 (K12, ara. Δ (lac-pro), thi/F′, pro A+B+,laclqZΔM15; Pharmacia Biotech).

Eukaryotic host cells are used in the second aspect of the invention.Yeast cells, mammalian cells such as, for example, CHO cells or humancells, insect cells or plant cells are preferably used.

The process according to the invention can also be employed forobtaining recombinant S-layer proteins. This is done by using a nucleicacid coding for the S-layer protein and comprising one or moreinsertions which code for peptide or polypeptide sequences. Theseinsertions may, on the one hand, code only for peptides with a few aminoacids, for example 1-25 amino acids. On the other hand, the insertionsmay also code for larger polypeptides of, for example, up to 1000 aminoacids and preferably up to 500 amino acids, without the S-layer proteinlosing the ability to form a correctly folded structure. Besides theinsertions, the recombinant S-layer protein may also comprise amino acidsubstitutions, in particular substitutions of single amino acids in theregion of the insertion site, and, where appropriate, deletions ofsingle amino acids or short amino acid sections of up to 30 amino acids.

Preferred insertion sites for peptide- or polypeptide-encoding sequencesin the sbsA gene are regions between positions 200-3600 of thenucleotide sequence shown in SEQ ID NO. 1. Particularly preferredinsertion sites are the NruI cleavage site at position 585,the PvuIIcleavage site at position 881,the SnaB I cleavage site at position920,the PvuII cleavage site at position 2507 and the PvuII cleavage siteat position 2652 (PCT/EP 97/00 432). Further preferred insertion sitesare positions 562, 1087, 1813, 1947, 2295, 2652, 3046, 3484 and 3594.The positions stated in each case relate to the first nucleotide of theinsertion.

Preferred insertion sites into the sbsB gene are regions betweenpositions 200-2600 of the nucleotide sequence shown in SEQ ID NO. 5.Particularly preferred insertion sites are positions 410 (codon 136),484 (codon 161/162) and 1583 (codon 528/529) (PCT/EP 97/00432). Furtherpreferred insertion sites are positions 598, 1012, 1435, 1808 and2301,the position indicated in each case relating to the firstnucleotide of the insertion.

The peptide- or polypeptide-encoding insertions are preferably selectedfrom nucleotide sequences which code for cysteine residues, regions withseveral charged amino acids, for example Arg, Lys, Asp or Glu, or Tyrresidues, DNA-binding epitopes, antigenic, allergenic or immunogenicepitopes, metal-binding epitopes, streptavidin, enzymes, cytokines orantibody-binding proteins.

A particularly preferred example of an insertion into the nucleic acidcoding for the S-layer protein is a nucleotide sequence coding forstreptavidin. It is possible in this way to obtain universal carriermolecules which are suitable for coupling biotinylated reagents to theintegrated streptavidin of the recombinant S-layer protein and fordetection in immunological or hybridization test methods.

Another preferred example of insertions comprises antigenic, allergenicor immunogenic epitopes, for example epitopes from pathogenicmicroorganisms such as, for example, bacteria, fungi, parasites etc. andviruses, or epitopes from plants or epitopes against endogenoussubstances, for example cytokines, and against toxins, in particularendotoxins. Particularly preferred examples of immunogenic epitopes areepitopes from viruses, for example from herpesviruses such as, forexample herpesvirus 1,for example glycoprotein Δ, herpesvirus 6 orpseudorabiesvirus (Lomniczi et al., J. Virol. 49 (1984), 970-979), inparticular epitopes from the gB, gC or/and gD genes, epitopes from footand mouth disease virus (FMDV), in particular epitopes from the genesections which code for VP1, VP2 or/and VP3, epitopes from flavivirusesor epitopes from filoviruses such as, for example, Ebola, Marburg orLassa virus. The immunogenic epitopes can be selected so that theypromote the generation of an antibody-mediated immune response or/andpromote the generation of a cellular immune response, for example bystimulating T cells. Examples of suitable allergenic epitopes are birchpollen allergens, for example Bet v I (Ebner et al., J. Immunol. 150(1993) 1047-1054). Also particularly preferred are antigenic epitopesable to bind and filter out, from serum or other body fluids, endogenousor exogenous substances such as, for example, cytokines or toxins.Epitopes of this type may comprise constituents of cytokine receptors ortoxin receptors or of antibodies against cytokines or toxins.

Modified S-layer proteins comprising immunogenic or/and antigenicepitopes with glycosylation sites are preferably produced in eukaryotichost cells in which glycosylation is possible. It is also possible inthis case for the natural S-layer sequences to be glycosylated. Examplesof potential N-glycosylation sites in the S-layer gene sbsA are aminoacid positions 26, 285, 343, 387, 388, 418, 421, 483, 653, 675, 902,924, 1048, 1058, 1118, 1154 and 1161. A potential N-glycosylation maytake place in the sbsB gene at positions 155, 184, 213, 302, 303, 400,463, 606, 755 and 915. Further possible modifications of the sbsA genecomprise amidation, phosphorylation by casein kinase II,N-myristoylation and phosphorylation by protein kinase C. Furtherpossible modifications of the sbsB gene comprise phosphorylation bycAMP- and cGMP-dependent protein kinase, phosphorylation by caseinkinase II, N-myristoylation, phosphorylation by protein kinase C andattachment to a fibronectin receptor (via sequence RGD).

On the other hand, the insertions may also code for enymzes. Preferredexamples are enzymes for synthe-sizing polyhydroxybutyric acid, forexample PHB synthase. Incorporation of PHB synthase into the S-layer mayproduce, on addition of the substrate hydroxybutyric acid under suitableconditions, a molecular spinneret. Another preferred example of anenzyme is bacterial luciferase. In this case, a molecular laser can beobtained on addition of the enzyme substrate, an aldehyde, and FMNH₂(reduced flavin mononucleotide), and in the presence of O₂.

There is likewise preference for insertions which code for cytokinessuch as, for example, interleukins, interferons or tumor necrosisfactors. These molecules can be used, for example, in combination withimmunogenic epitopes for producing vaccines.

Finally, there is also preference for insertions which code forantibody-binding proteins such as, for example, protein A or protein Gor for DNA- or/and metal-binding epitopes such as, for example, leucinezippers, zinc fingers etc.

Thus, the present invention provides for the first time a Gram-negativeprokaryotic cell which comprises immobilized recombinant polypeptides innative form, for example active enzymes, in the outer membrane, in thecytoplasmic membrane, preferably on the inside thereof or/and in theperiplasm. It is possible in this way for 50,000-200,000,for exampleabout 100,000, recombinant molecules to be immobilized per mm² ofrecombinant S-layer. Up to 3000 m² of S-layer can be obtained per kg ofrecombinant E.coli cells.

The present invention further provides for the first time a eukaryoticcell which comprises immobilized recombinant S-layer polypeptides in thecytoplasmic membrane, preferably on the inside thereof or/and in cellorganelles such as, for example, the Golgi apparatus, lysosomes,mitochondria, chloroplasts, vacuoles or endoplasmic reticulum.

Preference is further given, in particular for secretion into theperiplasm, to recombinant S-layer proteins into which cysteine residueshave been incorporated. It is possible, by a selection of the insertionpositions, to achieve covalent crosslinking of the S-layers in theperiplasm or/and on insertion at positions unsuitable for crosslinkingit is possible to provide docking sites for polypeptides, for examplefor enzymes, which can be covalently linked via a free SH group to theS-layer. Suitable and particularly preferred for this purpose arerecombinant polypeptides into which an additional cysteine residue hasbeen introduced by genetic manipulation methods, preferably at the N orC terminus or at a domain localized on the surface and which, throughselection of a suitable expression system, are likewise secreted intothe periplasm of the recombinant host cell.

In the process according to the invention, the nucleic acid coding forthe S-layer protein is used operatively linked to a nucleic acid codingfor a signal peptide of Gram-negative bacteria or of eukaryotic cells,i.e. the signal peptide-encoding nucleic acid is located on the 5′ sideof the S-layer protein-encoding nucleic acid.

On integration into the outer membrane of prokaryotic Gram-negative hostcells, the C-terminal domain of the IgA protease from neisseria orhaemophilus (Klauser et al., J. Mor Bio. 234 (1993), 579-593) can beused as signal peptide-encoding sequence.

On integration into the cytoplasmic membrane of Gram-negativeprokaryotic host cells it is preferable to use a hydrophobicmembrane-integrating protein domain which has no lytic activity and hasan α-helical structure. Examples of DNA sequences which code for such amembrane-integrating protein domain are described in European patent 0516 655.

On secretion into the periplasm of Gram-negative prokaryotic cells it ispossible for the nucleic acid coding for the signal peptide to comprise(a) the signal peptide-encoding section of the nucleotide sequencedepicted in SEQ ID NO. 7 and FIG. 4, (b) a nucleotide sequencecorresponding to the sequence from (a) within the scope of thedegeneracy of the genetic code or/and (c) a nucleotide sequence which isat least 80% and, in particular, at least 90% homologous with thesequences from (a) or/and (b). Other sequences which bring aboutsecretion into the periplasm are described, for example, by Blondel andBedouelle (Eur. J. Biochem 193 (1990), 325-330; Adip-Conquy et al.(Protein Eng. 8 (1995), 859-863); Weller et al (Eur. J. Biochem. 236(1996), 34-39) and Dubreuil et al. (FEMA Immunol. Med. Microbiol. 13(1996), 317-323).

On secretion into the extracellular medium of Gram-negative prokaryoticcells it is possible for the nucleic acid coding for the signal peptideto comprise (a) the signal peptide-encoding section of the nucleotidesequence depicted in SEQ ID NO. 8 and FIG. 5, (b) a nucleotide sequencecorresponding to the sequence from (a) within the scope of thedegeneracy of the genetic code or/and (c) a nucleotide sequence which isat least 80% and, in particular, at least 90% homologous with thesequences from (a) or/and (b). However, other signal peptide-encodingsequences are also suitable in addition, as described, for example, byYuan et al. (Appl. Environ. Microbiol. 63 (1997), 263-269) andHoogenboom et al. (Nucleic Acids Res. 19 (1991), 4133-4137).

Signal peptide-encoding nucleic acids known for expression in thecytoplasmic membrane or in organelles of eukaryotic cells are theN-terminal transit peptide of plastocyanin for transport in chloroplasts(Weisbeek et al., J. Cell. Sci. Suppl. 11 (1989), 199-223),mitochondrial signal peptides for transport in mitochondria (Skerjanc,Biochem. Cell. Biol. 68 (1990), 9-16), targeting sequences for transportin vacuoles (Vitale and Chrispeels, Bioessays 14 (1992), 151-160),targeting sequences for the cell membrane, the cytoplasm and the Golgiapparatus (Stanley, Mol. Membr. Biol. 13 (1996), 19-27), retentionsignals for the endoplasmic reticulum (Lencer et al., J. Cell. Biol. 131(1995), 951-962) and transfer sequences for the Golgi apparatus or theplasma membrane (Rambourg et al., Anat. Rec. 245 (1996), 447-458).

Signal peptide-encoding nucleic acids known for secretion into theextracellular medium of eukaryotic cells are the hsp 150 delta carrier(Jamsa et al., Yeast 11 (1995), 1381-1391), the signal peptide ofmelittin from the honeybee (Sisk et al., J. Virol 68 (1994), 766-775),signal peptides from baculovirus (Murphy et al., Protein Expr. Purif. 4(1993), 349-357), fragments of the K1 killer preprotoxin (Cartwright etal., Yeast 8 (1992), 261-272), the signal peptide and the N-terminalproregion of peptidylglycine α-hydroxylating monooxygenase (Mains etal., Mol. Endocrinol. 9 (1995), 3-13), the maltose-binding protein MalEwith its signal sequence (Staropoli et al., J. Virol. Methods 56 (1996),179-189;Clement and Jehanna, J. Biotechnol. 43 (1995), 169-181), theprepro-α-factor leader region of the yeast MF α1 gene (Elliot et al.,Gene 79 (1989), 167-180), the signal sequence of the IL-1 receptorantagonist (Wingren et al., Cell Immunol. 169 (1996), 226-237), thesignal peptide of the wheat α-amylase gene (Ribbe and Nagarajan, Mol.Gen. Genet. 235 (1992), 333-339), secretion polypeptides from fungi(Punt et al., Antonio Van Leeuwenhoek 65 (1994), 211-216), the leaderpeptide of the killer toxin from Kluyveromyces lactis (Baldari et al.,EMBO J. 6 (1987), 229-234) and the inulinase signal sequence (Kang etal., J. Biotechnol. 48 (1996), 15-24). Fusion constructs from MalE andSbsA and from MalE and SbsB are described in the present application.

Besides the section coding for the signal peptide, the DNA sequencecoding for the S-layer protein may comprise one or more other sectionswhich code for other protein domains. Such a section may preferably belocated between the section coding for the signal peptide and thesection coding for the S-layer protein. This section preferably codesfor a secretory polypeptide from Gram-negative bacteria or eukaryoticorganisms or a part thereof. A preferred example of such a nucleic acidsection is the malE gene which encodes the maltose-binding protein.

In a preferred embodiment of the process according to the invention, itis also possible to express several S-layer genes in a single host cell.For this purpose there is preferably expression of at least two S-layergenes, in which case one bf them codes for a modified S-layer proteinand another codes for an unmodified S-layer protein. The unmodifiedS-layer protein is preferably able to form an S-layer structure which iscompatible with the modified S-layer protein. One example of thisembodiment of the process according to the invention is an E.coli cellwhich is transformed with two S-layer genes, one of which is a naturalsbsA or sbsB gene and the other is a recombinant sbsA or sbsB gene.

The present invention further relates to a nucleic acid which codes foran S-layer protein optionally comprising heterologous peptide orpolypeptide insertions and is operatively linked to a signal sequencewhich codes for a peptide which brings about

(a) integration into the outer or cytoplasmic membrane of aGram-negative prokaryotic host cell, secretion into the periplasmicspace of a Gram-negative prokaryotic host cell or/and secretion into theextra-cellular medium of a Gram-negative prokaryotic host cell, or

(b) integration into the cytoplasmic membrane of a eukaryotic host cell,integration into an organelle of a eukaryotic host cell or/and secretioninto the extracellular medium of a eukaryotic host cell.

The nucleic acid preferably codes for a recombinant S-layer protein asindicated above.

The present invention further relates to a recombinant vector whichcomprises at least one copy of a nucleic acid according to theinvention. The vector is preferably replicable in prokaryotes or/and ineukaryotes. The vector is particularly preferably a prokaryotic oreukaryotic plasmid. It is further preferred for the vector to be thenucleic acid according to the invention operatively linked to anexpression control sequence which is active in Gram-negative oreukaryotic cells. The expression control sequence particularlypreferably comprises a regulable promoter. Examples of suitableprokaryotic promoters are the tac, lac, trp or λ promoter. Examples ofsuitable eukaryotic promoters are the SV40, CMV or metallothioneinpromoter.

The present invention further relates also to a host cell which istransformed with a nucleic acid or a recombinant vector according to thepresent invention. The cell is preferably a Gram-negative prokaryoticcell, for example an E.coli cell, or a eukaryotic cell, for example ayeast cell or a CHO cell. The cell according to the invention maycomprise a recombinant S-layer structure in the cytoplasmic membrane,the periplasm or a cell organelle. Processes for the transformation ofcells with nucleic acids are general prior art (see Sambrook et al.,supra) and therefore need not be explained.

A recombinant S-layer structure comprising as subunit at least onerecombinant S-layer protein according to the invention can be assembledfrom recombinant S-layer protein molecules. It is further preferred forthe S-layer structure according to the invention also to compriseunmodified S-layer proteins as “diluting molecules”. The unmodifiedS-layer proteins are preferably present in a molar proportion of 10-99%based on the total S-layer proteins.

The S-layer structure according to the invention may comprise severallayers which are linked together covalently or by affinity binding.Covalent linkages can be introduced, for example, by insertions ofcysteine residues and a subsequent formation of cystine bridges.Linkages by affinity binding comprise, for example, antibody-antigen,antibody-protein A or antibody-protein G or streptavidin-biotininteractions.

S-layer structures comprising recombinant S-layer proteins may also beproduced where appropriate in carrier-bound form. This can be done byreassembling the S-layer structure from individual units in the presenceof a peptidoglycan carrier, producing, for example, peptidoglycan layerswhich are covered on one or both sides with an S-layer structure.Another possibility for producing carrier-bound S-layer structures is toproduce a layer of S-layers at an interface between two media, forexample water/air, and to immobilize this layer on a solid phase, forexample a filter membrane (cf., for example, Pum and Sleytr (1994), ThinSolid Films 244, 882-886; Küpcü et al. (1995), Biochim. Biophys. Acta1235, 263-269).

The recombinant S-layer proteins and S-layer structures are suitable fora large number of applications. A particularly preferred use is asvaccine or adjuvant, in which case the recombinant S-layer proteins usedcomprise immunogenic epitopes of pathogens and/or endogenousimmunostimulant polypeptides such as, for example, cytokines.Purification of the recombinant S-layer proteins is not absolutelynecessary for this application. It is possible instead to use, forexample, a combination with a bacterial ghost which comprises additionalimmunogenic epitopes where appropriate in its periplasmic space, itsouter membrane or its cytoplasmic membrane.

The production of suitable “bacterial ghosts” is described, for example,in the International Patent Application PCT/EP91/00967, to whichreference is made herewith. This discloses modified bacteria obtainableby transformation of a Gram-negative bacterium with the gene of amembrane protein having lytic activity from bacteriophages, with thegene of a toxin-release protein having lytic activity or with geneswhich comprise part-sequences thereof which code for lytic proteins,cultivation of the bacterium, expression of this lysis gene andisolation of the resulting bacterial ghost from the culture medium.

A recombinant protein which is obtainable by expression of a recombinantDNA in these Gram-negative bacteria can be bound to the membrane ofthese bacteria as described in European Patent 0 516 655. Thisrecombinant DNA comprises a first DNA sequence which codes for ahydrophobic membrane-integrating protein domain which has no lyticactivity, has an α-helical structure and consists of 14-20 amino acidswhich may be flanked N- and C-terminally by, in each case, 2-30 suitableamino acids. A second DNA sequence which codes for a requiredrecombinant protein is operatively linked to this first DNA sequence.The Gram-negative bacterium additionally comprises a third DNA sequencewhich is subject to a control separate from the first and second DNAsequences and codes for a membrane protein having lytic activity frombacteriophages or a toxin-release protein having lytic activity or forthe parts thereof having lytic activity. So-called “bacterial ghosts”are obtained by expression and lysis of such recombinant Gram-negativebacteria and comprise an intact surface structure with immunogenicepitopes bound to the surface.

On combination of these bacterial ghosts with recombinant S-layersaccording to the invention it is possible to produce vaccines andadjuvants which have particularly advantageous properties.

Another particularly preferred use of recombinant S-layer proteins andS-layer structures is the use as enzyme reactor. Such an enzyme reactorcan be formed, for example, by a cell which comprises in its interior arecombinant S-layer structure according to the invention. On the otherhand, the enzyme reactor may also be formed from isolated S-layerstructures which have been reassembled in vitro, or combinations ofvarious S-layer structures.

The present invention is further illustrated by the following examplesand figures. These show:

SEQ ID NO. 1 the complete nucleotide sequence of the coding section ofthe S-layer gene sbsA of B.stearothermophilus;

SEQ ID NO. 2 the amino acid sequence derived therefrom;

SEQ ID NO. 3 the nucleotide sequence of the primer T5-X;

SEQ ID NO. 4 the nucleotide sequence of the primer E;

SEQ ID NO. 5 the complete nucleotide sequence of the coding section ofthe S-layer gene sbsB of B.stearothermophilus;

SEQ ID NO. 6 the amino acid sequence derived therefrom;

SEQ ID NO. 7 the signal sequence of the malE gene;

SEQ ID NO. 8 the signal sequence of gene 3 of bacteriophage fd;

FIG. 1 a diagrammatic representation of the sbsA PCR fragment used toproduce the recombinant vector pBK4;

FIG. 2 a diagrammatic representation of the production of the vectorpMAL-A comprising the malE-sbsA fusion gene (Example 7),

FIG. 3 a diagrammatic representation of the vector pCant-A (Example 8),

FIG. 4 the nucleotide sequence of an sbsA gene fused to the malE geneincluding its signal sequence,

FIG. 5 the nucleotide sequence of an sbsA gene fused to the signalsequence of gene 3 of bacteriophage fd and

FIG. 6 the nucleotide sequence of an sbsB gene fused to the malE geneincluding its signal sequence.

EXAMPLES 1. Bacterial Strains, Media and Plasmids

Gram-positive bacteria of the strain Bacillus stearothermophilus PV72were cultivated in SVIII medium (Bartelmus and Perschak. Z. Zuckerind.,78 (1957), 276-281) at 58° C. E.coli bacteria were cultivated in LBmedium (Sambrook et al., (1989), supra). To select transformants,ampicillin was added to the medium in a final concentration of 100μg/ml. The plasmid pPLcAT10 (λpl, bla, colE1) (Stanssens et al., Gene 36(1985), 211-223) was used as cloning vector.

2. Manipulation of DNA Fragments

Restriction analysis of DNA, agarose gel electrophoresis and cloning ofDNA fragments were carried out by the standard methods described bySambrook et al. (1989), supra.

The transformation of competent cells took place by electroporationusing a Bio-Rad gene pulser (Bio-Rad Laboratories, Richmond, Calif.,USA) in accordance with the manufacturer's protocols.

Plasmid DNA was isolated by the method of Birnboim and Doly (NucleicAcids Res. 7 (1979), 1513-1523). Chromosomal DNA was isolated by themethods described by Ausubel et al. (Current Protocols in MolecularBiology (1987), New York, John Wiley).

Restriction endonucleases and other enzymes were purchased fromBoehringer Mannheim, New England Biolabs or Strategene and were employedin accordance with the manufacturer's instructions.

3. DNA Sequencing

Sequence analysis of DNA molecules took place by the dideoxychain-termination method of Sanger et al. The primers used forsequencing the sbsA gene were constructed on the basis of the sbsAsequence which had already been published (Kuen et al., Gene 145 (1994),115-120).

4. PCR Amplification of sbsA

PCR amplification of the sbsA gene took place in a reaction volume of100 μl which contained 200 μM deoxynucleotides, 1 U of Pfu polymerase(Strategene), 1×Pfu reaction buffer, 0.5 μM respective oligonucleotideprimers and 100 ng of genomic DNA from B.stearothermophilus as template.The amplification was carried out over 30 cycles in a thermocycler(Biomed Thermocycler 60). Each cycle consisted of a denaturation step at95° C. for 1.5 min, an annealing step at 56° C. for 1 min and at 50° C.for 1 min and an extension step at 72° C. for 2 min.

The primers used were the primer T5-X which is indicated in the sequencelisting as SEQ ID NO. 3 and which flanks the 5′ region of sbsA andcomprises an XbaI site, and the primer E which is shown in the sequencelisting in SEQ ID NO. 4 and which flanks the region, located 20nucleotides downstream, of the transcription terminator of the sbsAsequence and comprises a BamHI site.

The PCR-amplified products were fractionated by electrophoresis on a0.8% agarose gel and purified for the cloning by using the Gene Cleansystem (BIO101 La Jolla, Calif., USA).

5. Cloning of the sbsA Gene into the Vector pPLcAT10

The sbsA gene with a length of 3.79 kb obtained by PCR was purified andcleaved with the restriction endonucleases XbaI and BamHI. The resultingXBaI-BamHI fragment was cloned into the corresponding restriction sitesof the vector pPLcAT10 so that the sbsA gene was under thetranscriptional control of the pL promoter located upstream. The ATGstart codon of the sbsA sequence was reconstructed by the cloningprocedure. The cloned sbsA sequence comprised the N-terminal signalsequence of sbsA and terminated 20 nt after the transcriptionterminator. After ligation of the vector DNA with the sbsA fragment, theE.coli strain pop2135 (DSM 10509) was transformed byelectrotransformation. The resulting clones were subjected to a DNArestriction analysis. One positive clone was sequenced in order toverify the correct sequence junctions at the 5′ and 3′ ends. This clonewas called pBK4.

A diagrammatic representation of the 3.79 kb XbaI sbsA fragment and itslocation in the multiple cloning site of the plasmid pBK4 is depicted inFIG. 1 (abbreviations: tT: transcription terminator; ori: origin of DNAreplication; amp: ampicillin-resistance gene).

6. Recombinant Expression of the sbsA Gene in the Cytoplasm of E.coli(Comparative Example)

E.coli pop2135/pBK4 cells were cultivated at 28° C. until the opticaldensity OD₆₀₀ reached 0.3. The expression of sbsA was then induced byincreasing the cultivation temperature from 28° C. to 42°C. 1.5 mlaliquots were taken before and 1, 2, 3 and 5 hours after induction ofsbsA expression. The controls used were E.coli pop2135/pPLcAT10(cultivated under the same conditions) and B.stearothermophilus PV72.

Culture supernatants and cell extracts from all the samples wereinvestigated for expression of the S-layer protein by SDS-PAGE andWestern immunoblotting.

For the Western blot, the proteins were transferred to a nitrocellulosemembrane and incubated with a rabbit polyclonal antiserum against SbsA.The production of this antiserum is described by Egelseer et al. (J.Bacteriol. 177 (1995), 1444-1451). A conjugate of goat anti-rabbit IgGand alkaline phosphatase was used to detect bound SbsA-specificantibodies.

An additional strong protein band with approximately the same molecularweight as the wild-type SbsA protein was found in cytoplasmic extractsfrom E.coli cells transformed with pBK4.

No SbsA protein was detectable in supernatants of E.coli cellstransformed with pBK4, even after induction of sbsA gene expression. Itis evident from this that SbsA is not exported into the surroundingmedium.

7. Secretion of the SbsA Protein into the Periplasm

The sbsA gene was cloned without signal sequence and with stop codon atthe 3′ end into the polylinker of the commercially available plasmidpMAL-P2 (New England Biolabs) (FIG. 2). The resulting plasmid pMAL-Acomprises, under the control of the taq promoter, a fusion genecomprising the malE gene including its signal sequence, and the sbsAgene without its signal sequence. A factor Xa cleavage site is locatedbetween the two domains.

Analysis of the crude extract of E.coli DH5α cells (Hanahan (1983)supra) transformed with pMAL-A showed expression of a MalE-SbsA fusionpolypeptide with a molecular weight of about 170 kDa in the periplasmicfraction, which was produced by a cold osmotic shock procedure (Neu andHeppel, J. Biol. Chem. 240 (1965); 3685-2692), of the cell extract. Thenucleotide sequence of the malE-sbsA fusion gene is depicted in FIG. 4.The malE signal sequence is shown in SEQ ID NO. 7.

8. Secretion of the SbsA Protein into the Extracellular Space

The plasmid pCant-A was produced by cloning the sbsA gene without itsown signal sequence and with stop codon at the 3′ end into thecommercially available plasmid pCANTAB5E (Pharmacia Biotech) which hadbeen cut with SfiI and NotI. It comprises, under the control of the lacpromoter (Plac), the signal sequence of gene 3 of bacteriophage fd (45nt) fused to the sbsA gene without its own signal sequence (FIG. 3). Thenucleotide sequence of the fusion gene is depicted in FIG. 5. The fdgene 3 signal sequence is shown in SEQ ID NO. 8.

The SbsA protein was detectable in the culture supernatant from E.coliHB2151 cells (Pharmacia Biotech) transformed with pCant-A.

9. Secretion of the SbsB Protein into the Periplasm and into theExtracellular Space

The sbsB gene was cloned, as described in Examples 7 and 8,without itsown signal sequence, into the plasmids pMAL-P2 and pCANTAB5E, resultingin the plasmids pMAL-8 and pCant-B.

Secretion of the sbsB protein into the periplasm and into theextracellular space was demonstrable in E.coli cells transformed withthe plasmids pMAL-B and pCant-B.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 13 <210> SEQ ID NO 1 <211>LENGTH: 3687 <212> TYPE: DNA <213> ORGANISM: Bacillus stearothermophilus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(3684) <221>NAME/KEY: sig_peptide <222> LOCATION: (1)..(90) <221> NAME/KEY:mat_peptide <222> LOCATION: (91)..(3684) <400> SEQUENCE: 1 atg gat aggaaa aaa gct gtg aaa cta gca aca gca agt gct att gca 48 Met Asp Arg LysLys Ala Val Lys Leu Ala Thr Ala Ser Ala Ile Ala -30 -25 -20 -15 gca agtgca ttt gtc gct gca aat cca aac gct tct gaa gcg gct aca 96 Ala Ser AlaPhe Val Ala Ala Asn Pro Asn Ala Ser Glu Ala Ala Thr -10 -5 -1 1 gat gtagca aca gta gta agc caa gca aaa gca cag ttc aaa aaa gca 144 Asp Val AlaThr Val Val Ser Gln Ala Lys Ala Gln Phe Lys Lys Ala 5 10 15 tac tat acttac agc cat aca gta acg gaa act ggt gaa ttc cca aac 192 Tyr Tyr Thr TyrSer His Thr Val Thr Glu Thr Gly Glu Phe Pro Asn 20 25 30 att aac gat gtatat gct gaa tac aac aaa gcg aaa aaa cga tac cgt 240 Ile Asn Asp Val TyrAla Glu Tyr Asn Lys Ala Lys Lys Arg Tyr Arg 35 40 45 50 gat gcg gta gcatta gtg aat aaa gca ggt ggc gcg aaa aaa gac gct 288 Asp Ala Val Ala LeuVal Asn Lys Ala Gly Gly Ala Lys Lys Asp Ala 55 60 65 tac tta gct gat ttacaa aaa gaa tat gaa act tac gtt ttc aaa gca 336 Tyr Leu Ala Asp Leu GlnLys Glu Tyr Glu Thr Tyr Val Phe Lys Ala 70 75 80 aac cct aaa tct ggc gaagct cgt gta gca act tac atc gat gct tac 384 Asn Pro Lys Ser Gly Glu AlaArg Val Ala Thr Tyr Ile Asp Ala Tyr 85 90 95 aac tat gca aca aaa tta gacgaa atg cgc caa gag cta gag gct gct 432 Asn Tyr Ala Thr Lys Leu Asp GluMet Arg Gln Glu Leu Glu Ala Ala 100 105 110 gtt caa gca aaa gat tta gaaaaa gca gaa caa tac tat cac aaa att 480 Val Gln Ala Lys Asp Leu Glu LysAla Glu Gln Tyr Tyr His Lys Ile 115 120 125 130 cct tat gaa att aaa actcgc aca gtc att tta gat cgc gta tat ggt 528 Pro Tyr Glu Ile Lys Thr ArgThr Val Ile Leu Asp Arg Val Tyr Gly 135 140 145 aaa aca act cgt gat ttactt cgc tct aca ttt aaa gca aaa gca caa 576 Lys Thr Thr Arg Asp Leu LeuArg Ser Thr Phe Lys Ala Lys Ala Gln 150 155 160 gaa ctt cgc gac agc ttaatt tat gat att acc gtt gca atg aaa gcg 624 Glu Leu Arg Asp Ser Leu IleTyr Asp Ile Thr Val Ala Met Lys Ala 165 170 175 cgc gaa gta caa gac gctgtg aaa gca ggc aat tta gac aaa gct aaa 672 Arg Glu Val Gln Asp Ala ValLys Ala Gly Asn Leu Asp Lys Ala Lys 180 185 190 gct gct gtt gat caa atcaat caa tac tta cca aaa gta aca gat gct 720 Ala Ala Val Asp Gln Ile AsnGln Tyr Leu Pro Lys Val Thr Asp Ala 195 200 205 210 ttc aaa act gaa ctaaca gaa gta gcg aaa aaa gca tta gat gca gat 768 Phe Lys Thr Glu Leu ThrGlu Val Ala Lys Lys Ala Leu Asp Ala Asp 215 220 225 gaa gct gcg ctt actcca aaa gtt gaa agt gta agt gcg att aac act 816 Glu Ala Ala Leu Thr ProLys Val Glu Ser Val Ser Ala Ile Asn Thr 230 235 240 caa aac aaa gct gttgaa tta aca gca gta cca gtg aac gga aca cta 864 Gln Asn Lys Ala Val GluLeu Thr Ala Val Pro Val Asn Gly Thr Leu 245 250 255 aaa tta caa ctt tcagct gct gca aat gaa gat aca gta aac gta aat 912 Lys Leu Gln Leu Ser AlaAla Ala Asn Glu Asp Thr Val Asn Val Asn 260 265 270 act gta cgt atc tataaa gtg gac ggt aac att cca ttt gcc ctt aat 960 Thr Val Arg Ile Tyr LysVal Asp Gly Asn Ile Pro Phe Ala Leu Asn 275 280 285 290 acg gca gat gtttct tta tct aca gac gga aaa act atc act gtg gat 1008 Thr Ala Asp Val SerLeu Ser Thr Asp Gly Lys Thr Ile Thr Val Asp 295 300 305 gct tca act ccattc gaa aat aat acg gag tat aaa gta gta gtt aaa 1056 Ala Ser Thr Pro PheGlu Asn Asn Thr Glu Tyr Lys Val Val Val Lys 310 315 320 ggt att aaa gacaaa aat ggc aaa gaa ttt aaa gaa gat gca ttc act 1104 Gly Ile Lys Asp LysAsn Gly Lys Glu Phe Lys Glu Asp Ala Phe Thr 325 330 335 ttc aag ctt cgaaat gat gct gta gtt act caa gtg ttt gga act aat 1152 Phe Lys Leu Arg AsnAsp Ala Val Val Thr Gln Val Phe Gly Thr Asn 340 345 350 gta aca aac aacact tct gta aac tta gca gca ggt act ttc gac act 1200 Val Thr Asn Asn ThrSer Val Asn Leu Ala Ala Gly Thr Phe Asp Thr 355 360 365 370 gac gat acttta aca gta gta ttt gat aag ttg tta gca cct gaa act 1248 Asp Asp Thr LeuThr Val Val Phe Asp Lys Leu Leu Ala Pro Glu Thr 375 380 385 gta aac agctcg aac gtt act att aca gat gtt gaa act gga aaa cgc 1296 Val Asn Ser SerAsn Val Thr Ile Thr Asp Val Glu Thr Gly Lys Arg 390 395 400 att cca gtaatt gca tct act tct ggt tct aca att act att acg tta 1344 Ile Pro Val IleAla Ser Thr Ser Gly Ser Thr Ile Thr Ile Thr Leu 405 410 415 aaa gaa gcgtta gta act ggt aaa caa tat aaa ctt gct atc aat aat 1392 Lys Glu Ala LeuVal Thr Gly Lys Gln Tyr Lys Leu Ala Ile Asn Asn 420 425 430 gtt aaa acatta act ggt tac aat gca gaa gct tac gag tta gtg ttc 1440 Val Lys Thr LeuThr Gly Tyr Asn Ala Glu Ala Tyr Glu Leu Val Phe 435 440 445 450 act gcaaac gca tca gca cca act gtt gct acc gct cct act act tta 1488 Thr Ala AsnAla Ser Ala Pro Thr Val Ala Thr Ala Pro Thr Thr Leu 455 460 465 ggt ggtaca act tta tct act ggt tct ctt aca aca aat gtt tgg ggt 1536 Gly Gly ThrThr Leu Ser Thr Gly Ser Leu Thr Thr Asn Val Trp Gly 470 475 480 aaa ttggct ggt ggt gtg aat gaa gct gga act tat tat cct ggt ctt 1584 Lys Leu AlaGly Gly Val Asn Glu Ala Gly Thr Tyr Tyr Pro Gly Leu 485 490 495 caa ttcaca aca acg ttt gct act aag tta gac gaa tct act tta gct 1632 Gln Phe ThrThr Thr Phe Ala Thr Lys Leu Asp Glu Ser Thr Leu Ala 500 505 510 gat aacttt gta tta gtt gaa aaa gaa tct ggt aca gtt gtt gct tct 1680 Asp Asn PheVal Leu Val Glu Lys Glu Ser Gly Thr Val Val Ala Ser 515 520 525 530 gaacta aaa tat aat gca gac gct aaa atg gta act tta gtg cca aaa 1728 Glu LeuLys Tyr Asn Ala Asp Ala Lys Met Val Thr Leu Val Pro Lys 535 540 545 gcggac ctt aaa gaa aat aca atc tat caa atc aaa att aaa aaa ggc 1776 Ala AspLeu Lys Glu Asn Thr Ile Tyr Gln Ile Lys Ile Lys Lys Gly 550 555 560 ttgaag tcc gat aaa ggt att gaa tta ggc act gtt aac gag aaa aca 1824 Leu LysSer Asp Lys Gly Ile Glu Leu Gly Thr Val Asn Glu Lys Thr 565 570 575 tatgag ttc aaa act caa gac tta act gct cct aca gtt att agc gta 1872 Tyr GluPhe Lys Thr Gln Asp Leu Thr Ala Pro Thr Val Ile Ser Val 580 585 590 acgtct aaa aat ggc gac gct gga tta aaa gta act gaa gct caa gaa 1920 Thr SerLys Asn Gly Asp Ala Gly Leu Lys Val Thr Glu Ala Gln Glu 595 600 605 610ttt act gtg aag ttc tca gag aat tta aat aca ttt aat gct aca acc 1968 PheThr Val Lys Phe Ser Glu Asn Leu Asn Thr Phe Asn Ala Thr Thr 615 620 625gtt tcg ggt agc aca atc aca tac ggt caa gtt gct gta gta aaa gcg 2016 ValSer Gly Ser Thr Ile Thr Tyr Gly Gln Val Ala Val Val Lys Ala 630 635 640ggt gca aac tta tct gct ctt aca gca agt gac atc att cca gct agt 2064 GlyAla Asn Leu Ser Ala Leu Thr Ala Ser Asp Ile Ile Pro Ala Ser 645 650 655gtt gaa gcg gtt act ggt caa gat gga aca tac aaa gtg aaa gtt gct 2112 ValGlu Ala Val Thr Gly Gln Asp Gly Thr Tyr Lys Val Lys Val Ala 660 665 670gct aac caa tta gaa cgt aac caa ggg tac aaa tta gta gtg ttc ggt 2160 AlaAsn Gln Leu Glu Arg Asn Gln Gly Tyr Lys Leu Val Val Phe Gly 675 680 685690 aaa ggt gca aca gct cct gtt aaa gat gct gca aat gca aat act tta 2208Lys Gly Ala Thr Ala Pro Val Lys Asp Ala Ala Asn Ala Asn Thr Leu 695 700705 gca act aac tat atc tat aca ttt aca act gaa ggt caa gac gta aca 2256Ala Thr Asn Tyr Ile Tyr Thr Phe Thr Thr Glu Gly Gln Asp Val Thr 710 715720 gca cca acg gtt aca aaa gta ttc aaa ggt gat tct tta aaa gac gct 2304Ala Pro Thr Val Thr Lys Val Phe Lys Gly Asp Ser Leu Lys Asp Ala 725 730735 gat gca gtt act aca ctt acg aac gtt gat gca ggt caa aaa ttc act 2352Asp Ala Val Thr Thr Leu Thr Asn Val Asp Ala Gly Gln Lys Phe Thr 740 745750 atc caa ttt agc gaa gaa tta aaa act tct agt ggt tct tta gtg ggt 2400Ile Gln Phe Ser Glu Glu Leu Lys Thr Ser Ser Gly Ser Leu Val Gly 755 760765 770 ggc aaa gta act gtc gag aaa tta aca aac aac gga tgg gta gat gct2448 Gly Lys Val Thr Val Glu Lys Leu Thr Asn Asn Gly Trp Val Asp Ala 775780 785 ggt act gga aca act gta tca gtt gct cct aag aca gat gca aat ggt2496 Gly Thr Gly Thr Thr Val Ser Val Ala Pro Lys Thr Asp Ala Asn Gly 790795 800 aaa gta aca gct gct gtg gtt aca tta act ggt ctt gac aat aac gac2544 Lys Val Thr Ala Ala Val Val Thr Leu Thr Gly Leu Asp Asn Asn Asp 805810 815 aaa gat gcg aaa ttg cgt ctg gta gta gat aag tct tct act gat gga2592 Lys Asp Ala Lys Leu Arg Leu Val Val Asp Lys Ser Ser Thr Asp Gly 820825 830 att gct gat gta gct ggt aat gta att aag gaa aaa gat att tta att2640 Ile Ala Asp Val Ala Gly Asn Val Ile Lys Glu Lys Asp Ile Leu Ile 835840 845 850 cgt tac aac agc tgg aga cac act gta gct tct gtg aaa gct gctgct 2688 Arg Tyr Asn Ser Trp Arg His Thr Val Ala Ser Val Lys Ala Ala Ala855 860 865 gac aaa gat ggt caa aac gct tct gct gca ttc cca aca agc actgca 2736 Asp Lys Asp Gly Gln Asn Ala Ser Ala Ala Phe Pro Thr Ser Thr Ala870 875 880 att gat aca act aag agc tta tta gtt gaa ttc aat gaa act gattta 2784 Ile Asp Thr Thr Lys Ser Leu Leu Val Glu Phe Asn Glu Thr Asp Leu885 890 895 gcg gaa gtt aaa cct gag aac atc gtt gtt aaa gat gca gca ggtaat 2832 Ala Glu Val Lys Pro Glu Asn Ile Val Val Lys Asp Ala Ala Gly Asn900 905 910 gcg gta gct ggt act gta aca gca tta gac ggt tct aca aat aaattt 2880 Ala Val Ala Gly Thr Val Thr Ala Leu Asp Gly Ser Thr Asn Lys Phe915 920 925 930 gta ttc act cca tct caa gaa tta aaa gct ggt aca gtt tactct gta 2928 Val Phe Thr Pro Ser Gln Glu Leu Lys Ala Gly Thr Val Tyr SerVal 935 940 945 aca att gac ggt gtg aga gat aaa gta ggt aac aca atc tctaaa tac 2976 Thr Ile Asp Gly Val Arg Asp Lys Val Gly Asn Thr Ile Ser LysTyr 950 955 960 att act tcg ttc aag act gta tct gcg aat cca acg tta tcttca atc 3024 Ile Thr Ser Phe Lys Thr Val Ser Ala Asn Pro Thr Leu Ser SerIle 965 970 975 agc att gct gac ggt gca gtt aac gtt gac cgt tct aaa acaatt aca 3072 Ser Ile Ala Asp Gly Ala Val Asn Val Asp Arg Ser Lys Thr IleThr 980 985 990 att gaa ttc agc gat tca gtt cca aac cca aca atc act cttaag aag 3120 Ile Glu Phe Ser Asp Ser Val Pro Asn Pro Thr Ile Thr Leu LysLys 995 1000 1005 1010 gct gac gga act tca ttt act aat tac act tta gtaaat gta aat aat 3168 Ala Asp Gly Thr Ser Phe Thr Asn Tyr Thr Leu Val AsnVal Asn Asn 1015 1020 1025 gaa aat aaa aca tac aaa att gta ttc cac aaaggt gta aca ctt gac 3216 Glu Asn Lys Thr Tyr Lys Ile Val Phe His Lys GlyVal Thr Leu Asp 1030 1035 1040 gag ttt act caa tat gag tta gca gtt tcaaaa gat ttt caa act ggt 3264 Glu Phe Thr Gln Tyr Glu Leu Ala Val Ser LysAsp Phe Gln Thr Gly 1045 1050 1055 act gat att gat agc aaa gtt aca ttcatc aca ggt tct gtt gct act 3312 Thr Asp Ile Asp Ser Lys Val Thr Phe IleThr Gly Ser Val Ala Thr 1060 1065 1070 gac gaa gta aaa cct gct cta gtaggc gtt ggt tca tgg aat gga aca 3360 Asp Glu Val Lys Pro Ala Leu Val GlyVal Gly Ser Trp Asn Gly Thr 1075 1080 1085 1090 agc tat act cag gat gctgca gca aca cga ctt cgg tct gta gct gac 3408 Ser Tyr Thr Gln Asp Ala AlaAla Thr Arg Leu Arg Ser Val Ala Asp 1095 1100 1105 ttc gtt gcg gag ccagtt gcc ctt caa ttc tca gaa ggt atc gat tta 3456 Phe Val Ala Glu Pro ValAla Leu Gln Phe Ser Glu Gly Ile Asp Leu 1110 1115 1120 acg aat gca actgtg aca gta aca aat att act gat gat aaa act gtt 3504 Thr Asn Ala Thr ValThr Val Thr Asn Ile Thr Asp Asp Lys Thr Val 1125 1130 1135 gaa gtt atttca aaa gag agt gta gac gca gac cat gat gca ggt gct 3552 Glu Val Ile SerLys Glu Ser Val Asp Ala Asp His Asp Ala Gly Ala 1140 1145 1150 act aaggag aca tta gta att aac aca gtt act cct tta gta ctt gat 3600 Thr Lys GluThr Leu Val Ile Asn Thr Val Thr Pro Leu Val Leu Asp 1155 1160 1165 1170aac agc aag act tat aag att gtt gta agt gga gtt aaa gat gca gca 3648 AsnSer Lys Thr Tyr Lys Ile Val Val Ser Gly Val Lys Asp Ala Ala 1175 11801185 ggt aat gtt gca gat act att aca ttc tat att aag taa 3687 Gly AsnVal Ala Asp Thr Ile Thr Phe Tyr Ile Lys 1190 1195 <210> SEQ ID NO 2<211> LENGTH: 1228 <212> TYPE: PRT <213> ORGANISM: Bacillusstearothermophilus <400> SEQUENCE: 2 Met Asp Arg Lys Lys Ala Val Lys LeuAla Thr Ala Ser Ala Ile Ala -30 -25 -20 -15 Ala Ser Ala Phe Val Ala AlaAsn Pro Asn Ala Ser Glu Ala Ala Thr -10 -5 -1 1 Asp Val Ala Thr Val ValSer Gln Ala Lys Ala Gln Phe Lys Lys Ala 5 10 15 Tyr Tyr Thr Tyr Ser HisThr Val Thr Glu Thr Gly Glu Phe Pro Asn 20 25 30 Ile Asn Asp Val Tyr AlaGlu Tyr Asn Lys Ala Lys Lys Arg Tyr Arg 35 40 45 50 Asp Ala Val Ala LeuVal Asn Lys Ala Gly Gly Ala Lys Lys Asp Ala 55 60 65 Tyr Leu Ala Asp LeuGln Lys Glu Tyr Glu Thr Tyr Val Phe Lys Ala 70 75 80 Asn Pro Lys Ser GlyGlu Ala Arg Val Ala Thr Tyr Ile Asp Ala Tyr 85 90 95 Asn Tyr Ala Thr LysLeu Asp Glu Met Arg Gln Glu Leu Glu Ala Ala 100 105 110 Val Gln Ala LysAsp Leu Glu Lys Ala Glu Gln Tyr Tyr His Lys Ile 115 120 125 130 Pro TyrGlu Ile Lys Thr Arg Thr Val Ile Leu Asp Arg Val Tyr Gly 135 140 145 LysThr Thr Arg Asp Leu Leu Arg Ser Thr Phe Lys Ala Lys Ala Gln 150 155 160Glu Leu Arg Asp Ser Leu Ile Tyr Asp Ile Thr Val Ala Met Lys Ala 165 170175 Arg Glu Val Gln Asp Ala Val Lys Ala Gly Asn Leu Asp Lys Ala Lys 180185 190 Ala Ala Val Asp Gln Ile Asn Gln Tyr Leu Pro Lys Val Thr Asp Ala195 200 205 210 Phe Lys Thr Glu Leu Thr Glu Val Ala Lys Lys Ala Leu AspAla Asp 215 220 225 Glu Ala Ala Leu Thr Pro Lys Val Glu Ser Val Ser AlaIle Asn Thr 230 235 240 Gln Asn Lys Ala Val Glu Leu Thr Ala Val Pro ValAsn Gly Thr Leu 245 250 255 Lys Leu Gln Leu Ser Ala Ala Ala Asn Glu AspThr Val Asn Val Asn 260 265 270 Thr Val Arg Ile Tyr Lys Val Asp Gly AsnIle Pro Phe Ala Leu Asn 275 280 285 290 Thr Ala Asp Val Ser Leu Ser ThrAsp Gly Lys Thr Ile Thr Val Asp 295 300 305 Ala Ser Thr Pro Phe Glu AsnAsn Thr Glu Tyr Lys Val Val Val Lys 310 315 320 Gly Ile Lys Asp Lys AsnGly Lys Glu Phe Lys Glu Asp Ala Phe Thr 325 330 335 Phe Lys Leu Arg AsnAsp Ala Val Val Thr Gln Val Phe Gly Thr Asn 340 345 350 Val Thr Asn AsnThr Ser Val Asn Leu Ala Ala Gly Thr Phe Asp Thr 355 360 365 370 Asp AspThr Leu Thr Val Val Phe Asp Lys Leu Leu Ala Pro Glu Thr 375 380 385 ValAsn Ser Ser Asn Val Thr Ile Thr Asp Val Glu Thr Gly Lys Arg 390 395 400Ile Pro Val Ile Ala Ser Thr Ser Gly Ser Thr Ile Thr Ile Thr Leu 405 410415 Lys Glu Ala Leu Val Thr Gly Lys Gln Tyr Lys Leu Ala Ile Asn Asn 420425 430 Val Lys Thr Leu Thr Gly Tyr Asn Ala Glu Ala Tyr Glu Leu Val Phe435 440 445 450 Thr Ala Asn Ala Ser Ala Pro Thr Val Ala Thr Ala Pro ThrThr Leu 455 460 465 Gly Gly Thr Thr Leu Ser Thr Gly Ser Leu Thr Thr AsnVal Trp Gly 470 475 480 Lys Leu Ala Gly Gly Val Asn Glu Ala Gly Thr TyrTyr Pro Gly Leu 485 490 495 Gln Phe Thr Thr Thr Phe Ala Thr Lys Leu AspGlu Ser Thr Leu Ala 500 505 510 Asp Asn Phe Val Leu Val Glu Lys Glu SerGly Thr Val Val Ala Ser 515 520 525 530 Glu Leu Lys Tyr Asn Ala Asp AlaLys Met Val Thr Leu Val Pro Lys 535 540 545 Ala Asp Leu Lys Glu Asn ThrIle Tyr Gln Ile Lys Ile Lys Lys Gly 550 555 560 Leu Lys Ser Asp Lys GlyIle Glu Leu Gly Thr Val Asn Glu Lys Thr 565 570 575 Tyr Glu Phe Lys ThrGln Asp Leu Thr Ala Pro Thr Val Ile Ser Val 580 585 590 Thr Ser Lys AsnGly Asp Ala Gly Leu Lys Val Thr Glu Ala Gln Glu 595 600 605 610 Phe ThrVal Lys Phe Ser Glu Asn Leu Asn Thr Phe Asn Ala Thr Thr 615 620 625 ValSer Gly Ser Thr Ile Thr Tyr Gly Gln Val Ala Val Val Lys Ala 630 635 640Gly Ala Asn Leu Ser Ala Leu Thr Ala Ser Asp Ile Ile Pro Ala Ser 645 650655 Val Glu Ala Val Thr Gly Gln Asp Gly Thr Tyr Lys Val Lys Val Ala 660665 670 Ala Asn Gln Leu Glu Arg Asn Gln Gly Tyr Lys Leu Val Val Phe Gly675 680 685 690 Lys Gly Ala Thr Ala Pro Val Lys Asp Ala Ala Asn Ala AsnThr Leu 695 700 705 Ala Thr Asn Tyr Ile Tyr Thr Phe Thr Thr Glu Gly GlnAsp Val Thr 710 715 720 Ala Pro Thr Val Thr Lys Val Phe Lys Gly Asp SerLeu Lys Asp Ala 725 730 735 Asp Ala Val Thr Thr Leu Thr Asn Val Asp AlaGly Gln Lys Phe Thr 740 745 750 Ile Gln Phe Ser Glu Glu Leu Lys Thr SerSer Gly Ser Leu Val Gly 755 760 765 770 Gly Lys Val Thr Val Glu Lys LeuThr Asn Asn Gly Trp Val Asp Ala 775 780 785 Gly Thr Gly Thr Thr Val SerVal Ala Pro Lys Thr Asp Ala Asn Gly 790 795 800 Lys Val Thr Ala Ala ValVal Thr Leu Thr Gly Leu Asp Asn Asn Asp 805 810 815 Lys Asp Ala Lys LeuArg Leu Val Val Asp Lys Ser Ser Thr Asp Gly 820 825 830 Ile Ala Asp ValAla Gly Asn Val Ile Lys Glu Lys Asp Ile Leu Ile 835 840 845 850 Arg TyrAsn Ser Trp Arg His Thr Val Ala Ser Val Lys Ala Ala Ala 855 860 865 AspLys Asp Gly Gln Asn Ala Ser Ala Ala Phe Pro Thr Ser Thr Ala 870 875 880Ile Asp Thr Thr Lys Ser Leu Leu Val Glu Phe Asn Glu Thr Asp Leu 885 890895 Ala Glu Val Lys Pro Glu Asn Ile Val Val Lys Asp Ala Ala Gly Asn 900905 910 Ala Val Ala Gly Thr Val Thr Ala Leu Asp Gly Ser Thr Asn Lys Phe915 920 925 930 Val Phe Thr Pro Ser Gln Glu Leu Lys Ala Gly Thr Val TyrSer Val 935 940 945 Thr Ile Asp Gly Val Arg Asp Lys Val Gly Asn Thr IleSer Lys Tyr 950 955 960 Ile Thr Ser Phe Lys Thr Val Ser Ala Asn Pro ThrLeu Ser Ser Ile 965 970 975 Ser Ile Ala Asp Gly Ala Val Asn Val Asp ArgSer Lys Thr Ile Thr 980 985 990 Ile Glu Phe Ser Asp Ser Val Pro Asn ProThr Ile Thr Leu Lys Lys 995 1000 1005 1010 Ala Asp Gly Thr Ser Phe ThrAsn Tyr Thr Leu Val Asn Val Asn Asn 1015 1020 1025 Glu Asn Lys Thr TyrLys Ile Val Phe His Lys Gly Val Thr Leu Asp 1030 1035 1040 Glu Phe ThrGln Tyr Glu Leu Ala Val Ser Lys Asp Phe Gln Thr Gly 1045 1050 1055 ThrAsp Ile Asp Ser Lys Val Thr Phe Ile Thr Gly Ser Val Ala Thr 1060 10651070 Asp Glu Val Lys Pro Ala Leu Val Gly Val Gly Ser Trp Asn Gly Thr1075 1080 1085 1090 Ser Tyr Thr Gln Asp Ala Ala Ala Thr Arg Leu Arg SerVal Ala Asp 1095 1100 1105 Phe Val Ala Glu Pro Val Ala Leu Gln Phe SerGlu Gly Ile Asp Leu 1110 1115 1120 Thr Asn Ala Thr Val Thr Val Thr AsnIle Thr Asp Asp Lys Thr Val 1125 1130 1135 Glu Val Ile Ser Lys Glu SerVal Asp Ala Asp His Asp Ala Gly Ala 1140 1145 1150 Thr Lys Glu Thr LeuVal Ile Asn Thr Val Thr Pro Leu Val Leu Asp 1155 1160 1165 1170 Asn SerLys Thr Tyr Lys Ile Val Val Ser Gly Val Lys Asp Ala Ala 1175 1180 1185Gly Asn Val Ala Asp Thr Ile Thr Phe Tyr Ile Lys 1190 1195 <210> SEQ IDNO 3 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Primer T5-X <400> SEQUENCE: 3 ttaatcgatt ctagatggataggaaaaaag ctg 33 <210> SEQ ID NO 4 <211> LENGTH: 37 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primer E <400>SEQUENCE: 4 atacccgggg gtacggatcc gatacagatt tgagcaa 37 <210> SEQ ID NO5 <211> LENGTH: 2763 <212> TYPE: DNA <213> ORGANISM: Bacillusstearothermophilus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(2760) <221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(93) <221>NAME/KEY: mat_peptide <222> LOCATION: (94)..(2760) <400> SEQUENCE: 5 atggct tat caa cct aag tcc tat cgc aag ttt gtt gcg aca act gca 48 Met AlaTyr Gln Pro Lys Ser Tyr Arg Lys Phe Val Ala Thr Thr Ala -30 -25 -20 acagct gcc atg gta gca tct gcg gta gct cct gta gta tct gca gca 96 Thr AlaAla Met Val Ala Ser Ala Val Ala Pro Val Val Ser Ala Ala -15 -10 -5 -1 1agc ttc aca gat gtt gcg ccg caa tat aaa gat gcg atc gat ttc tta 144 SerPhe Thr Asp Val Ala Pro Gln Tyr Lys Asp Ala Ile Asp Phe Leu 5 10 15 gtatca act ggt gca aca aaa ggt aaa aca gaa aca aaa ttc ggc gtt 192 Val SerThr Gly Ala Thr Lys Gly Lys Thr Glu Thr Lys Phe Gly Val 20 25 30 tac gatgaa atc act cgt cta gat gcg gca gtt att ctt gca aga gta 240 Tyr Asp GluIle Thr Arg Leu Asp Ala Ala Val Ile Leu Ala Arg Val 35 40 45 tta aaa ctagac gtt gac aac gca aaa gac gca ggc ttc aca gat gtg 288 Leu Lys Leu AspVal Asp Asn Ala Lys Asp Ala Gly Phe Thr Asp Val 50 55 60 65 cca aaa gaccgt gca aaa tac gtc aac gcg ctt gta gaa gct ggc gta 336 Pro Lys Asp ArgAla Lys Tyr Val Asn Ala Leu Val Glu Ala Gly Val 70 75 80 tta aac ggt aaagca cct ggc aaa ttt ggt gca tac gac cca tta act 384 Leu Asn Gly Lys AlaPro Gly Lys Phe Gly Ala Tyr Asp Pro Leu Thr 85 90 95 cgc gtt gaa atg gcaaaa atc atc gcg aac cgt tac aaa tta aaa gct 432 Arg Val Glu Met Ala LysIle Ile Ala Asn Arg Tyr Lys Leu Lys Ala 100 105 110 gac gat gta aaa cttcca ttc act gat gta aac gat aca tgg gca cca 480 Asp Asp Val Lys Leu ProPhe Thr Asp Val Asn Asp Thr Trp Ala Pro 115 120 125 tac gta aaa gcg ctttat aaa tac gaa gta aca aaa ggt aaa aca cca 528 Tyr Val Lys Ala Leu TyrLys Tyr Glu Val Thr Lys Gly Lys Thr Pro 130 135 140 145 aca agc ttc ggtgca tac caa aac atc act cgc ggt gac ttt gcg caa 576 Thr Ser Phe Gly AlaTyr Gln Asn Ile Thr Arg Gly Asp Phe Ala Gln 150 155 160 ttt gta tat agagcg gtg aat att aat gca gtg cca gaa ata gtt gaa 624 Phe Val Tyr Arg AlaVal Asn Ile Asn Ala Val Pro Glu Ile Val Glu 165 170 175 gta act gcg gttaat tcg act aca gtg aaa gta aca ttc aat acg caa 672 Val Thr Ala Val AsnSer Thr Thr Val Lys Val Thr Phe Asn Thr Gln 180 185 190 att gct gat gttgat ttc aca aat ttt gct atc gat aac ggt tta act 720 Ile Ala Asp Val AspPhe Thr Asn Phe Ala Ile Asp Asn Gly Leu Thr 195 200 205 gtt act aaa gcaact ctt tct cgt gat aaa aaa tcc gta gag gtt gtg 768 Val Thr Lys Ala ThrLeu Ser Arg Asp Lys Lys Ser Val Glu Val Val 210 215 220 225 gta aat aaaccg ttt act cgt aat cag gaa tat aca att aca gcg aca 816 Val Asn Lys ProPhe Thr Arg Asn Gln Glu Tyr Thr Ile Thr Ala Thr 230 235 240 ggc att aaaaat tta aaa ggc gag acc gct aag gaa tta act ggt aag 864 Gly Ile Lys AsnLeu Lys Gly Glu Thr Ala Lys Glu Leu Thr Gly Lys 245 250 255 ttt gtt tggtct gtt caa gat gcg gta act gtt gca cta aat aat agt 912 Phe Val Trp SerVal Gln Asp Ala Val Thr Val Ala Leu Asn Asn Ser 260 265 270 tcg ctt aaagtt gga gag gaa tct ggt tta act gta aaa gat cag gat 960 Ser Leu Lys ValGly Glu Glu Ser Gly Leu Thr Val Lys Asp Gln Asp 275 280 285 ggc aaa gatgtt gta ggt gct aaa gta gaa ctt act tct tct aat act 1008 Gly Lys Asp ValVal Gly Ala Lys Val Glu Leu Thr Ser Ser Asn Thr 290 295 300 305 aat attgtt gta gtt tca agt ggc gaa gta tca gta tct gct gct aaa 1056 Asn Ile ValVal Val Ser Ser Gly Glu Val Ser Val Ser Ala Ala Lys 310 315 320 gtt acagct gta aaa ccg gga aca gct gat gtt act gca aaa gtt aca 1104 Val Thr AlaVal Lys Pro Gly Thr Ala Asp Val Thr Ala Lys Val Thr 325 330 335 tta ccagat ggt gtt gta cta aca aat aca ttt aaa gtg aca gtt aca 1152 Leu Pro AspGly Val Val Leu Thr Asn Thr Phe Lys Val Thr Val Thr 340 345 350 gaa gtgcct gtg caa gta caa aat caa gga ttt act tta gtt gat aat 1200 Glu Val ProVal Gln Val Gln Asn Gln Gly Phe Thr Leu Val Asp Asn 355 360 365 ctt tctaat gct cca cag aat aca gtt gca ttt aac aaa gct gag aaa 1248 Leu Ser AsnAla Pro Gln Asn Thr Val Ala Phe Asn Lys Ala Glu Lys 370 375 380 385 gtaact tca atg ttt gct gga gaa act aaa aca gtt gca atg tat gat 1296 Val ThrSer Met Phe Ala Gly Glu Thr Lys Thr Val Ala Met Tyr Asp 390 395 400 actaaa aac ggt gat cct gaa act aaa cct gtt gat ttc aaa gat gca 1344 Thr LysAsn Gly Asp Pro Glu Thr Lys Pro Val Asp Phe Lys Asp Ala 405 410 415 actgta cgt tca tta aat cca att att gca aca gct gct att aat ggt 1392 Thr ValArg Ser Leu Asn Pro Ile Ile Ala Thr Ala Ala Ile Asn Gly 420 425 430 agtgag ctc ctt gtc aca gct aat gct ggc caa tct gga aaa gct tca 1440 Ser GluLeu Leu Val Thr Ala Asn Ala Gly Gln Ser Gly Lys Ala Ser 435 440 445 tttgaa gta aca ttt aaa gat aat aca aaa aga aca ttt aca gtt gat 1488 Phe GluVal Thr Phe Lys Asp Asn Thr Lys Arg Thr Phe Thr Val Asp 450 455 460 465gtg aaa aaa gac cct gta tta caa gat att aaa gta gat gca act tct 1536 ValLys Lys Asp Pro Val Leu Gln Asp Ile Lys Val Asp Ala Thr Ser 470 475 480gtt aaa ctt tcc gat gaa gct gtt ggc ggc ggg gaa gtt gaa gga gtt 1584 ValLys Leu Ser Asp Glu Ala Val Gly Gly Gly Glu Val Glu Gly Val 485 490 495aac caa aaa acg att aaa gta agt gca gtt gac caa tac ggt aaa gaa 1632 AsnGln Lys Thr Ile Lys Val Ser Ala Val Asp Gln Tyr Gly Lys Glu 500 505 510att aaa ttt ggt aca aaa ggt aaa gtt act gtt aca act aat aca gaa 1680 IleLys Phe Gly Thr Lys Gly Lys Val Thr Val Thr Thr Asn Thr Glu 515 520 525gga cta gtt att aaa aat gta aat agc gat aat aca att gac ttt gat 1728 GlyLeu Val Ile Lys Asn Val Asn Ser Asp Asn Thr Ile Asp Phe Asp 530 535 540545 agc ggc aat agt gca act gac caa ttt gtt gtc gtt gca aca aaa gac 1776Ser Gly Asn Ser Ala Thr Asp Gln Phe Val Val Val Ala Thr Lys Asp 550 555560 aaa att gtc aat ggt aaa gta gaa gtt aaa tat ttc aaa aat gct agt 1824Lys Ile Val Asn Gly Lys Val Glu Val Lys Tyr Phe Lys Asn Ala Ser 565 570575 gac aca aca cca act tca act aaa aca att act gtt aat gta gtg aat 1872Asp Thr Thr Pro Thr Ser Thr Lys Thr Ile Thr Val Asn Val Val Asn 580 585590 gta aaa gct gac gct aca cca gta gga tta gat att gta gca cct tct 1920Val Lys Ala Asp Ala Thr Pro Val Gly Leu Asp Ile Val Ala Pro Ser 595 600605 gaa att gat gtg aat gct cca aac act gct tct act gca gat gtt gat 1968Glu Ile Asp Val Asn Ala Pro Asn Thr Ala Ser Thr Ala Asp Val Asp 610 615620 625 ttt att aat ttc gaa agt gtt gag att tat aca ctc gat tct aat ggt2016 Phe Ile Asn Phe Glu Ser Val Glu Ile Tyr Thr Leu Asp Ser Asn Gly 630635 640 aac cgt ctt aaa aaa gtt act cca act gca act aca ctt gta ggt act2064 Asn Arg Leu Lys Lys Val Thr Pro Thr Ala Thr Thr Leu Val Gly Thr 645650 655 aat gat tat gtt gaa gtt aat ggg aat gta tta caa ttc aag ggt aac2112 Asn Asp Tyr Val Glu Val Asn Gly Asn Val Leu Gln Phe Lys Gly Asn 660665 670 gat gaa tta acg cta tta act tct tct agt aca gta aac gtt gat gta2160 Asp Glu Leu Thr Leu Leu Thr Ser Ser Ser Thr Val Asn Val Asp Val 675680 685 aca gct gat gga att aca aaa cgt att cca gta aaa tat atc aac tct2208 Thr Ala Asp Gly Ile Thr Lys Arg Ile Pro Val Lys Tyr Ile Asn Ser 690695 700 705 gca agt gta cct gcc agt gca aca gta gca aca agt cct gtt actgtt 2256 Ala Ser Val Pro Ala Ser Ala Thr Val Ala Thr Ser Pro Val Thr Val710 715 720 aag ctt aat tca agt gat aat gat tta aca ttt gaa gaa tta atattc 2304 Lys Leu Asn Ser Ser Asp Asn Asp Leu Thr Phe Glu Glu Leu Ile Phe725 730 735 ggt gta att gac cct aca caa tta gtc aaa gat gaa gac atc aacgaa 2352 Gly Val Ile Asp Pro Thr Gln Leu Val Lys Asp Glu Asp Ile Asn Glu740 745 750 ttt att gca gtt tca aaa gcg gct aaa aat gat gga tat ttg tataat 2400 Phe Ile Ala Val Ser Lys Ala Ala Lys Asn Asp Gly Tyr Leu Tyr Asn755 760 765 aaa ccg ctt gta acg gtt aaa gat gca tca gga aaa gtt att ccaaca 2448 Lys Pro Leu Val Thr Val Lys Asp Ala Ser Gly Lys Val Ile Pro Thr770 775 780 785 ggt gca aat gtt tac ggt cta aat cat gat gca act aac ggaaac att 2496 Gly Ala Asn Val Tyr Gly Leu Asn His Asp Ala Thr Asn Gly AsnIle 790 795 800 tgg ttt gat gag gaa caa gct ggc tta gct aaa aaa ttt agtgat gta 2544 Trp Phe Asp Glu Glu Gln Ala Gly Leu Ala Lys Lys Phe Ser AspVal 805 810 815 cat ttt gat gtt gat ttt tca tta gct aac gtt gta aaa actggt agc 2592 His Phe Asp Val Asp Phe Ser Leu Ala Asn Val Val Lys Thr GlySer 820 825 830 ggt aca gtt tct tca tcg cca tca tta tct gac gca att caactt act 2640 Gly Thr Val Ser Ser Ser Pro Ser Leu Ser Asp Ala Ile Gln LeuThr 835 840 845 aat tca ggc gat gca gta tcg ttt aca tta gtt atc aaa tcaatt tat 2688 Asn Ser Gly Asp Ala Val Ser Phe Thr Leu Val Ile Lys Ser IleTyr 850 855 860 865 gtt aaa ggc gca gat aaa gat gat aat aac tta ctt gcagcc cct gtt 2736 Val Lys Gly Ala Asp Lys Asp Asp Asn Asn Leu Leu Ala AlaPro Val 870 875 880 tct gtc aat gtg act gtg aca aaa taa 2763 Ser Val AsnVal Thr Val Thr Lys 885 <210> SEQ ID NO 6 <211> LENGTH: 920 <212> TYPE:PRT <213> ORGANISM: Bacillus stearothermophilus <400> SEQUENCE: 6 MetAla Tyr Gln Pro Lys Ser Tyr Arg Lys Phe Val Ala Thr Thr Ala -30 -25 -20Thr Ala Ala Met Val Ala Ser Ala Val Ala Pro Val Val Ser Ala Ala -15 -10-5 -1 1 Ser Phe Thr Asp Val Ala Pro Gln Tyr Lys Asp Ala Ile Asp Phe Leu5 10 15 Val Ser Thr Gly Ala Thr Lys Gly Lys Thr Glu Thr Lys Phe Gly Val20 25 30 Tyr Asp Glu Ile Thr Arg Leu Asp Ala Ala Val Ile Leu Ala Arg Val35 40 45 Leu Lys Leu Asp Val Asp Asn Ala Lys Asp Ala Gly Phe Thr Asp Val50 55 60 65 Pro Lys Asp Arg Ala Lys Tyr Val Asn Ala Leu Val Glu Ala GlyVal 70 75 80 Leu Asn Gly Lys Ala Pro Gly Lys Phe Gly Ala Tyr Asp Pro LeuThr 85 90 95 Arg Val Glu Met Ala Lys Ile Ile Ala Asn Arg Tyr Lys Leu LysAla 100 105 110 Asp Asp Val Lys Leu Pro Phe Thr Asp Val Asn Asp Thr TrpAla Pro 115 120 125 Tyr Val Lys Ala Leu Tyr Lys Tyr Glu Val Thr Lys GlyLys Thr Pro 130 135 140 145 Thr Ser Phe Gly Ala Tyr Gln Asn Ile Thr ArgGly Asp Phe Ala Gln 150 155 160 Phe Val Tyr Arg Ala Val Asn Ile Asn AlaVal Pro Glu Ile Val Glu 165 170 175 Val Thr Ala Val Asn Ser Thr Thr ValLys Val Thr Phe Asn Thr Gln 180 185 190 Ile Ala Asp Val Asp Phe Thr AsnPhe Ala Ile Asp Asn Gly Leu Thr 195 200 205 Val Thr Lys Ala Thr Leu SerArg Asp Lys Lys Ser Val Glu Val Val 210 215 220 225 Val Asn Lys Pro PheThr Arg Asn Gln Glu Tyr Thr Ile Thr Ala Thr 230 235 240 Gly Ile Lys AsnLeu Lys Gly Glu Thr Ala Lys Glu Leu Thr Gly Lys 245 250 255 Phe Val TrpSer Val Gln Asp Ala Val Thr Val Ala Leu Asn Asn Ser 260 265 270 Ser LeuLys Val Gly Glu Glu Ser Gly Leu Thr Val Lys Asp Gln Asp 275 280 285 GlyLys Asp Val Val Gly Ala Lys Val Glu Leu Thr Ser Ser Asn Thr 290 295 300305 Asn Ile Val Val Val Ser Ser Gly Glu Val Ser Val Ser Ala Ala Lys 310315 320 Val Thr Ala Val Lys Pro Gly Thr Ala Asp Val Thr Ala Lys Val Thr325 330 335 Leu Pro Asp Gly Val Val Leu Thr Asn Thr Phe Lys Val Thr ValThr 340 345 350 Glu Val Pro Val Gln Val Gln Asn Gln Gly Phe Thr Leu ValAsp Asn 355 360 365 Leu Ser Asn Ala Pro Gln Asn Thr Val Ala Phe Asn LysAla Glu Lys 370 375 380 385 Val Thr Ser Met Phe Ala Gly Glu Thr Lys ThrVal Ala Met Tyr Asp 390 395 400 Thr Lys Asn Gly Asp Pro Glu Thr Lys ProVal Asp Phe Lys Asp Ala 405 410 415 Thr Val Arg Ser Leu Asn Pro Ile IleAla Thr Ala Ala Ile Asn Gly 420 425 430 Ser Glu Leu Leu Val Thr Ala AsnAla Gly Gln Ser Gly Lys Ala Ser 435 440 445 Phe Glu Val Thr Phe Lys AspAsn Thr Lys Arg Thr Phe Thr Val Asp 450 455 460 465 Val Lys Lys Asp ProVal Leu Gln Asp Ile Lys Val Asp Ala Thr Ser 470 475 480 Val Lys Leu SerAsp Glu Ala Val Gly Gly Gly Glu Val Glu Gly Val 485 490 495 Asn Gln LysThr Ile Lys Val Ser Ala Val Asp Gln Tyr Gly Lys Glu 500 505 510 Ile LysPhe Gly Thr Lys Gly Lys Val Thr Val Thr Thr Asn Thr Glu 515 520 525 GlyLeu Val Ile Lys Asn Val Asn Ser Asp Asn Thr Ile Asp Phe Asp 530 535 540545 Ser Gly Asn Ser Ala Thr Asp Gln Phe Val Val Val Ala Thr Lys Asp 550555 560 Lys Ile Val Asn Gly Lys Val Glu Val Lys Tyr Phe Lys Asn Ala Ser565 570 575 Asp Thr Thr Pro Thr Ser Thr Lys Thr Ile Thr Val Asn Val ValAsn 580 585 590 Val Lys Ala Asp Ala Thr Pro Val Gly Leu Asp Ile Val AlaPro Ser 595 600 605 Glu Ile Asp Val Asn Ala Pro Asn Thr Ala Ser Thr AlaAsp Val Asp 610 615 620 625 Phe Ile Asn Phe Glu Ser Val Glu Ile Tyr ThrLeu Asp Ser Asn Gly 630 635 640 Asn Arg Leu Lys Lys Val Thr Pro Thr AlaThr Thr Leu Val Gly Thr 645 650 655 Asn Asp Tyr Val Glu Val Asn Gly AsnVal Leu Gln Phe Lys Gly Asn 660 665 670 Asp Glu Leu Thr Leu Leu Thr SerSer Ser Thr Val Asn Val Asp Val 675 680 685 Thr Ala Asp Gly Ile Thr LysArg Ile Pro Val Lys Tyr Ile Asn Ser 690 695 700 705 Ala Ser Val Pro AlaSer Ala Thr Val Ala Thr Ser Pro Val Thr Val 710 715 720 Lys Leu Asn SerSer Asp Asn Asp Leu Thr Phe Glu Glu Leu Ile Phe 725 730 735 Gly Val IleAsp Pro Thr Gln Leu Val Lys Asp Glu Asp Ile Asn Glu 740 745 750 Phe IleAla Val Ser Lys Ala Ala Lys Asn Asp Gly Tyr Leu Tyr Asn 755 760 765 LysPro Leu Val Thr Val Lys Asp Ala Ser Gly Lys Val Ile Pro Thr 770 775 780785 Gly Ala Asn Val Tyr Gly Leu Asn His Asp Ala Thr Asn Gly Asn Ile 790795 800 Trp Phe Asp Glu Glu Gln Ala Gly Leu Ala Lys Lys Phe Ser Asp Val805 810 815 His Phe Asp Val Asp Phe Ser Leu Ala Asn Val Val Lys Thr GlySer 820 825 830 Gly Thr Val Ser Ser Ser Pro Ser Leu Ser Asp Ala Ile GlnLeu Thr 835 840 845 Asn Ser Gly Asp Ala Val Ser Phe Thr Leu Val Ile LysSer Ile Tyr 850 855 860 865 Val Lys Gly Ala Asp Lys Asp Asp Asn Asn LeuLeu Ala Ala Pro Val 870 875 880 Ser Val Asn Val Thr Val Thr Lys 885<210> SEQ ID NO 7 <211> LENGTH: 75 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: signal sequence of the malE gen <400> SEQUENCE:7 atgaaaataa aaacaggtgc acgcatcctc gcattatccg cattaacgac gatgatgttt 60tccgcctcgg ctctc 75 <210> SEQ ID NO 8 <211> LENGTH: 45 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: signal sequence of gen3of bacteriophage fd <400> SEQUENCE: 8 gtgaaaaaat tattattcgc aattcctttagttgttcctt tctat 45 <210> SEQ ID NO 9 <211> LENGTH: 49 <212> TYPE: DNA<213> ORGANISM: Bacillus stearothermophilus <400> SEQUENCE: 9 gaattcatcgatgtcgacca aggaggtcta gatggatccg gccaagctt 49 <210> SEQ ID NO 10 <211>LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:cloning strategy for pMAL-A <400> SEQUENCE: 10 atcgagggaa ggatttcagaattcggatcc tctagagtcg acctgcaggc aagcttg 57 <210> SEQ ID NO 11 <211>LENGTH: 4988 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:fusion of SbsA and MalE <400> SEQUENCE: 11 atgaaaataa aaacaggtgcacgcatcctc gcattatccg cattaacgac gatgatgttt 60 tccgcctcgg ctctcgccaaaatcgaagaa ggtaaactgg taatctggat taacggcgat 120 aaaggctata acggtctcgctgaagtcggt aagaaattcg agaaagatac cggaattaaa 180 gtcaccgttg agcatccggataaactggaa gagaaattcc cacaggttgc ggcaactggc 240 gatggccctg acattatcttctgggcacac gaccgctttg gtggctacgc tcaatctggc 300 ctgttggctg aaatcaccccggacaaagcg ttccaggaca agctgtatcc gtttacctgg 360 gatgccgtac gttacaacggcaagctgatt gcttacccga tcgctgttga agcgttatcg 420 ctgatttata acaaagatctgctgccgaac ccgccaaaaa cctgggaaga gatcccggcg 480 ctggataaag aactgaaagcgaaaggtaag agcgcgctga tgttcaacct gcaagaaccg 540 tacttcacct ggccgctgattgctgctgac gggggttatg cgttcaagta tgaaaacggc 600 aagtacgaca ttaaagacgtgggcgtggat aacgctggcg cgaaagcggg tctgaccttc 660 ctggttgacc tgattaaaaacaaacacatg aatgcagaca ccgattactc catcgcagaa 720 gctgccttta ataaaggcgaaacagcgatg accatcaacg gcccgtgggc atggtccaac 780 atcgacacca gcaaattgaattatggtgta acggtactgc cgaccttcaa gggtcaccca 840 tccaaaccgt tcgttggcgtgctgagcgca ggtattaacg ccgccagtcc gaacaaagag 900 ttggcgaaag agttcctcgaaaactatctg ctgactgatg aaggtctgga agcggttaat 960 aaagacaaac cgctgggtgccgtagcgctg aagtcttacg aggaagagtt ggcgaaagat 1020 ccacgtattg ccgccaccatggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg 1080 cagatgtccg ctttctggtatgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt 1140 cagatcgtcg atgaagccctgaaagacgcg cagactaatt cgagctcgaa caacaacaac 1200 aataacaata acaacaacctcgggatcgag ggaaggattt cagaattcgg atccgctaca 1260 gatgtagcaa cagtagtaagccaagcaaaa gcacagttca aaaaagcata ctatacttac 1320 agccatacag taacggaaactggtgaattc ccaaacatta acgatgtata tgctgaatac 1380 aacaaagcga aaaaacgataccgtgatgcg gtagcattag tgaataaagc aggtggcgcg 1440 aaaaaagacg cttacttagctgatttacaa aaagaatatg aaacttacgt tttcaaagca 1500 aaccctaaat ctggcgaagctcgtgtagca acttacatcg atgcttacaa ctatgcaaca 1560 aaattagacg aaatgcgccaagagctagag gctgctgttc aagcaaaaga tttagaaaaa 1620 gcagaacaat actatcacaaaattccttat gaaattaaaa ctcgcacagt cattttagat 1680 cgcgtatatg gtaaaacaactcgtgattta cttcgctcta catttaaagc aaaagcacaa 1740 gaacttcgcg acagcttaatttatgatatt accgttgcaa tgaaagcgcg cgaagtacaa 1800 gacgctgtga aagcaggcaatttagacaaa gctaaagctg ctgttgatca aatcaatcaa 1860 tacttaccaa aagtaacagatgctttcaaa actgaactaa cagaagtagc gaaaaaagca 1920 ttagatgcag atgaagctgcgcttactcca aaagttgaaa gtgtaagtgc gattaacact 1980 caaaacaaag ctgttgaattaacagcagta ccagtgaacg gaacactaaa attacaactt 2040 tcagctgctg caaatgaagatacagtaaac gtaaatactg tacgtatcta taaagtggac 2100 ggtaacattc catttgcccttaatacggca gatgtttctt tatctacaga cggaaaaact 2160 atcactgtgg atgcttcaactccattcgaa aataatacgg agtataaagt agtagttaaa 2220 ggtattaaag acaaaaatggcaaagaattt aaagaagatg cattcacttt caagcttcga 2280 aatgatgctg tagttactcaagtgtttgga actaatgtaa caaacaacac ttctgtaaac 2340 ttagcagcag gtactttcgacactgacgat actttaacag tagtatttga taagttgtta 2400 gcacctgaaa ctgtaaacagctcgaacgtt actattacag atgttgaaac tggaaaacgc 2460 attccagtaa ttgcatctacttctggttct acaattacta ttacgttaaa agaagcgtta 2520 gtaactggta aacaatataaacttgctatc aataatgtta aaacattaac tggttacaat 2580 gcagaagctt acgagttagtgttcactgca aacgcatcag caccaactgt tgctaccgct 2640 cctactactt taggtggtacaactttatct actggttctc ttacaacaaa tgtttggggt 2700 aaattggctg gtggtgtgaatgaagctgga acttattatc ctggtcttca attcacaaca 2760 acgtttgcta ctaagttagacgaatctact ttagctgata actttgtatt agttgaaaaa 2820 gaatctggta cagttgttgcttctgaacta aaatataatg cagacgctaa aatggtaact 2880 ttagtgccaa aagcggaccttaaagaaaat acaatctatc aaatcaaaat taaaaaaggc 2940 ttgaagtccg ataaaggtattgaattaggc actgttaacg agaaaacata tgagttcaaa 3000 actcaagact taactgctcctacagttatt agcgtaacgt ctaaaaatgg cgacgctgga 3060 ttaaaagtaa ctgaagctcaagaatttact gtgaagttct cagagaattt aaatacattt 3120 aatgctacaa ccgtttcgggtagcacaatc acatacggtc aagttgctgt agtaaaagcg 3180 ggtgcaaact tatctgctcttacagcaagt gacatcattc cagctagtgt tgaagcggtt 3240 actggtcaag atggaacatacaaagtgaaa gttgctgcta accaattaga acgtaaccaa 3300 gggtacaaat tagtagtgttcggtaaaggt gcaacagctc ctgttaaaga tgctgcaaat 3360 gcaaatactt tagcaactaactatatctat acatttacaa ctgaaggtca agacgtaaca 3420 gcaccaacgg ttacaaaagtattcaaaggt gattctttaa aagacgctga tgcagttact 3480 acacttacga acgttgatgcaggtcaaaaa ttcactatcc aatttagcga agaattaaaa 3540 acttctagtg gttctttagtgggtggcaaa gtaactgtcg agaaattaac aaacaacgga 3600 tgggtagatg ctggtactggaacaactgta tcagttgctc ctaagacaga tgcaaatggt 3660 aaagtaacag ctgctgtggttacattaact ggtcttgaca ataacgacaa agatgcgaaa 3720 ttgcgtctgg tagtagataagtcttctact gatggaattg ctgatgtagc tggtaatgta 3780 attaaggaaa aagatattttaattcgttac aacagctgga gacacactgt agcttctgtg 3840 aaagctgctg ctgacaaagatggtcaaaac gcttctgctg cattcccaac aagcactgca 3900 attgatacaa ctaagagcttattagttgaa ttcaatgaaa ctgatttagc ggaagttaaa 3960 cctgagaaca tcgttgttaaagatgcagca ggtaatgcgg tagctggtac tgtaacagca 4020 ttagacggtt ctacaaataaatttgtattc actccatctc aagaattaaa agctggtaca 4080 gtttactctg taacaattgacggtgtgaga gataaagtag gtaacacaat ctctaaatac 4140 attacttcgt tcaagactgtatctgcgaat ccaacgttat cttcaatcag cattgctgac 4200 ggtgcagtta acgttgaccgttctaaaaca attacaattg aattcagcga ttcagttcca 4260 aacccaacaa tcactcttaagaaggctgac ggaacttcat ttactaatta cactttagta 4320 aatgtaaata atgaaaataaaacatacaaa attgtattcc acaaaggtgt aacacttgac 4380 gagtttactc aatatgagttagcagtttca aaagattttc aaactggtac tgatattgat 4440 agcaaagtta cattcatcacaggttctgtt gctactgacg aagtaaaacc tgctctagta 4500 ggcgttggtt catggaatggaacaagctat actcaggatg ctgcagcaac acgacttcgg 4560 tctgtagctg acttcgttgcggagccagtt gcccttcaat tctcagaagg tatcgattta 4620 acgaatgcaa ctgtgacagtaacaaatatt actgatgata aaactgttga agttatttca 4680 aaagagagtg tagacgcagaccatgatgca ggtgctacta aggagacatt agtaattaac 4740 acagttactc ctttagtacttgataacagc aagacttata agattgttgt aagtggagtt 4800 aaagatgcag caggtaatgttgcagatact attacattct atattaagta atctgggcta 4860 ggtgtttgtc accgctcaaggttgtcaaaa tatgtcgaaa agctctgcgg agagaaatct 4920 ctgcggggct tttctttttgctcaaatctg tatcaggatc ctctagagtc gacctgcagg 4980 caagcttg 4988 <210> SEQID NO 12 <211> LENGTH: 3768 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: fusion of SbsA with the signal sequenz of gen3 ofbacteriophage fd <400> SEQUENCE: 12 gtgaaaaaat tattattcgc aattcctttagttgttcctt tctatgcggc ccagccggcc 60 gctacagatg tagcaacagt agtaagccaagcaaaagcac agttcaaaaa agcatactat 120 acttacagcc atacagtaac ggaaactggtgaattcccaa acattaacga tgtatatgct 180 gaatacaaca aagcgaaaaa acgataccgtgatgcggtag cattagtgaa taaagcaggt 240 ggcgcgaaaa aagacgctta cttagctgatttacaaaaag aatatgaaac ttacgttttc 300 aaagcaaacc ctaaatctgg cgaagctcgtgtagcaactt acatcgatgc ttacaactat 360 gcaacaaaat tagacgaaat gcgccaagagctagaggctg ctgttcaagc aaaagattta 420 gaaaaagcag aacaatacta tcacaaaattccttatgaaa ttaaaactcg cacagtcatt 480 ttagatcgcg tatatggtaa aacaactcgtgatttacttc gctctacatt taaagcaaaa 540 gcacaagaac ttcgcgacag cttaatttatgatattaccg ttgcaatgaa agcgcgcgaa 600 gtacaagacg ctgtgaaagc aggcaatttagacaaagcta aagctgctgt tgatcaaatc 660 aatcaatact taccaaaagt aacagatgctttcaaaactg aactaacaga agtagcgaaa 720 aaagcattag atgcagatga agctgcgcttactccaaaag ttgaaagtgt aagtgcgatt 780 aacactcaaa acaaagctgt tgaattaacagcagtaccag tgaacggaac actaaaatta 840 caactttcag ctgctgcaaa tgaagatacagtaaacgtaa atactgtacg tatctataaa 900 gtggacggta acattccatt tgcccttaatacggcagatg tttctttatc tacagacgga 960 aaaactatca ctgtggatgc ttcaactccattcgaaaata atacggagta taaagtagta 1020 gttaaaggta ttaaagacaa aaatggcaaagaatttaaag aagatgcatt cactttcaag 1080 cttcgaaatg atgctgtagt tactcaagtgtttggaacta atgtaacaaa caacacttct 1140 gtaaacttag cagcaggtac tttcgacactgacgatactt taacagtagt atttgataag 1200 ttgttagcac ctgaaactgt aaacagctcgaacgttacta ttacagatgt tgaaactgga 1260 aaacgcattc cagtaattgc atctacttctggttctacaa ttactattac gttaaaagaa 1320 gcgttagtaa ctggtaaaca atataaacttgctatcaata atgttaaaac attaactggt 1380 tacaatgcag aagcttacga gttagtgttcactgcaaacg catcagcacc aactgttgct 1440 accgctccta ctactttagg tggtacaactttatctactg gttctcttac aacaaatgtt 1500 tggggtaaat tggctggtgg tgtgaatgaagctggaactt attatcctgg tcttcaattc 1560 acaacaacgt ttgctactaa gttagacgaatctactttag ctgataactt tgtattagtt 1620 gaaaaagaat ctggtacagt tgttgcttctgaactaaaat ataatgcaga cgctaaaatg 1680 gtaactttag tgccaaaagc ggaccttaaagaaaatacaa tctatcaaat caaaattaaa 1740 aaaggcttga agtccgataa aggtattgaattaggcactg ttaacgagaa aacatatgag 1800 ttcaaaactc aagacttaac tgctcctacagttattagcg taacgtctaa aaatggcgac 1860 gctggattaa aagtaactga agctcaagaatttactgtga agttctcaga gaatttaaat 1920 acatttaatg ctacaaccgt ttcgggtagcacaatcacat acggtcaagt tgctgtagta 1980 aaagcgggtg caaacttatc tgctcttacagcaagtgaca tcattccagc tagtgttgaa 2040 gcggttactg gtcaagatgg aacatacaaagtgaaagttg ctgctaacca attagaacgt 2100 aaccaagggt acaaattagt agtgttcggtaaaggtgcaa cagctcctgt taaagatgct 2160 gcaaatgcaa atactttagc aactaactatatctatacat ttacaactga aggtcaagac 2220 gtaacagcac caacggttac aaaagtattcaaaggtgatt ctttaaaaga cgctgatgca 2280 gttactacac ttacgaacgt tgatgcaggtcaaaaattca ctatccaatt tagcgaagaa 2340 ttaaaaactt ctagtggttc tttagtgggtggcaaagtaa ctgtcgagaa attaacaaac 2400 aacggatggg tagatgctgg tactggaacaactgtatcag ttgctcctaa gacagatgca 2460 aatggtaaag taacagctgc tgtggttacattaactggtc ttgacaataa cgacaaagat 2520 gcgaaattgc gtctggtagt agataagtcttctactgatg gaattgctga tgtagctggt 2580 aatgtaatta aggaaaaaga tattttaattcgttacaaca gctggagaca cactgtagct 2640 tctgtgaaag ctgctgctga caaagatggtcaaaacgctt ctgctgcatt cccaacaagc 2700 actgcaattg atacaactaa gagcttattagttgaattca atgaaactga tttagcggaa 2760 gttaaacctg agaacatcgt tgttaaagatgcagcaggta atgcggtagc tggtactgta 2820 acagcattag acggttctac aaataaatttgtattcactc catctcaaga attaaaagct 2880 ggtacagttt actctgtaac aattgacggtgtgagagata aagtaggtaa cacaatctct 2940 aaatacatta cttcgttcaa gactgtatctgcgaatccaa cgttatcttc aatcagcatt 3000 gctgacggtg cagttaacgt tgaccgttctaaaacaatta caattgaatt cagcgattca 3060 gttccaaacc caacaatcac tcttaagaaggctgacggaa cttcatttac taattacact 3120 ttagtaaatg taaataatga aaataaaacatacaaaattg tattccacaa aggtgtaaca 3180 cttgacgagt ttactcaata tgagttagcagtttcaaaag attttcaaac tggtactgat 3240 attgatagca aagttacatt catcacaggttctgttgcta ctgacgaagt aaaacctgct 3300 ctagtaggcg ttggttcatg gaatggaacaagctatactc aggatgctgc agcaacacga 3360 cttcggtctg tagctgactt cgttgcggagccagttgccc ttcaattctc agaaggtatc 3420 gatttaacga atgcaactgt gacagtaacaaatattactg atgataaaac tgttgaagtt 3480 atttcaaaag agagtgtaga cgcagaccatgatgcaggtg ctactaagga gacattagta 3540 attaacacag ttactccttt agtacttgataacagcaaga cttataagat tgttgtaagt 3600 ggagttaaag atgcagcagg taatgttgcagatactatta cattctatat taagtaatct 3660 gggctaggtg tttgtcaccg ctcaaggttgtcaaaatatg tcgaaaagct ctgcggagag 3720 aaatctctgc ggggcttttc tttttgctcaaatctgtatc gcggccgc 3768 <210> SEQ ID NO 13 <211> LENGTH: 4065 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: fusion of SbsB withMalE <400> SEQUENCE: 13 atgaaaataa aaacaggtgc acgcatcctc gcattatccgcattaacgac gatgatgttt 60 tccgcctcgg ctctcgccaa aatcgaagaa ggtaaactggtaatctggat taacggcgat 120 aaaggctata acggtctcgc tgaagtcggt aagaaattcgagaaagatac cggaattaaa 180 gtcaccgttg agcatccgga taaactggaa gagaaattcccacaggttgc ggcaactggc 240 gatggccctg acattatctt ctgggcacac gaccgctttggtggctacgc tcaatctggc 300 ctgttggctg aaatcacccc ggacaaagcg ttccaggacaagctgtatcc gtttacctgg 360 gatgccgtac gttacaacgg caagctgatt gcttacccgatcgctgttga agcgttatcg 420 ctgatttata acaaagatct gctgccgaac ccgccaaaaacctgggaaga gatcccggcg 480 ctggataaag aactgaaagc gaaaggtaag agcgcgctgatgttcaacct gcaagaaccg 540 tacttcacct ggccgctgat tgctgctgac gggggttatgcgttcaagta tgaaaacggc 600 aagtacgaca ttaaagacgt gggcgtggat aacgctggcgcgaaagcggg tctgaccttc 660 ctggttgacc tgattaaaaa caaacacatg aatgcagacaccgattactc catcgcagaa 720 gctgccttta ataaaggcga aacagcgatg accatcaacggcccgtgggc atggtccaac 780 atcgacacca gcaaattgaa ttatggtgta acggtactgccgaccttcaa gggtcaccca 840 tccaaaccgt tcgttggcgt gctgagcgca ggtattaacgccgccagtcc gaacaaagag 900 ttggcgaaag agttcctcga aaactatctg ctgactgatgaaggtctgga agcggttaat 960 aaagacaaac cgctgggtgc cgtagcgctg aagtcttacgaggaagagtt ggcgaaagat 1020 ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtgaaatcatgcc gaacatcccg 1080 cagatgtccg ctttctggta tgccgtgcgt actgcggtgatcaacgccgc cagcggtcgt 1140 cagatcgtcg atgaagccct gaaagacgcg cagactaattcgagctcgaa caacaacaac 1200 aataacaata acaacaacct cgggatcgag ggaaggatttcagaattcgg atccgcaagc 1260 ttcacagatg ttgcgccgca atataaagat gcgatcgatttcttagtatc aactggtgca 1320 acaaaaggta aaacagaaac aaaattcggc gtttacgatgaaatcactcg tctagatgcg 1380 gcagttattc ttgcaagagt attaaaacta gacgttgacaacgcaaaaga cgcaggcttc 1440 acagatgtgc caaaagaccg tgcaaaatac gtcaacgcgcttgtagaagc tggcgtatta 1500 aacggtaaag cacctggcaa atttggtgca tacgacccattaactcgcgt tgaaatggca 1560 aaaatcatcg cgaaccgtta caaattaaaa gctgacgatgtaaaacttcc attcactgat 1620 gtaaacgata catgggcacc atacgtaaaa gcgctttataaatacgaagt aacaaaaggt 1680 aaaacaccaa caagcttcgg tgcataccaa aacatcactcgcggtgactt tgcgcaattt 1740 gtatatagag cggtgaatat taatgcagtg ccagaaatagttgaagtaac tgcggttaat 1800 tcgactacag tgaaagtaac attcaatacg caaattgctgatgttgattt cacaaatttt 1860 gctatcgata acggtttaac tgttactaaa gcaactctttctcgtgataa aaaatccgta 1920 gaggttgtgg taaataaacc gtttactcgt aatcaggaatatacaattac agcgacaggc 1980 attaaaaatt taaaaggcga gaccgctaag gaattaactggtaagtttgt ttggtctgtt 2040 caagatgcgg taactgttgc actaaataat agttcgcttaaagttggaga ggaatctggt 2100 ttaactgtaa aagatcagga tggcaaagat gttgtaggtgctaaagtaga acttacttct 2160 tctaatacta atattgttgt agtttcaagt ggcgaagtatcagtatctgc tgctaaagtt 2220 acagctgtaa aaccgggaac agctgatgtt actgcaaaagttacattacc agatggtgtt 2280 gtactaacaa atacatttaa agtgacagtt acagaagtgcctgtgcaagt acaaaatcaa 2340 ggatttactt tagttgataa tctttctaat gctccacagaatacagttgc atttaacaaa 2400 gctgagaaag taacttcaat gtttgctgga gaaactaaaacagttgcaat gtatgatact 2460 aaaaacggtg atcctgaaac taaacctgtt gatttcaaagatgcaactgt acgttcatta 2520 aatccaatta ttgcaacagc tgctattaat ggtagtgagctccttgtcac agctaatgct 2580 ggccaatctg gaaaagcttc atttgaagta acatttaaagataatacaaa aagaacattt 2640 acagttgatg tgaaaaaaga ccctgtatta caagatattaaagtagatgc aacttctgtt 2700 aaactttccg atgaagctgt tggcggcggg gaagttgaaggagttaacca aaaaacgatt 2760 aaagtaagtg cagttgacca atacggtaaa gaaattaaatttggtacaaa aggtaaagtt 2820 actgttacaa ctaatacaga aggactagtt attaaaaatgtaaatagcga taatacaatt 2880 gactttgata gcggcaatag tgcaactgac caatttgttgtcgttgcaac aaaagacaaa 2940 attgtcaatg gtaaagtaga agttaaatat ttcaaaaatgctagtgacac aacaccaact 3000 tcaactaaaa caattactgt taatgtagtg aatgtaaaagctgacgctac accagtagga 3060 ttagatattg tagcaccttc tgaaattgat gtgaatgctccaaacactgc ttctactgca 3120 gatgttgatt ttattaattt cgaaagtgtt gagatttatacactcgattc taatggtaac 3180 cgtcttaaaa aagttactcc aactgcaact acacttgtaggtactaatga ttatgttgaa 3240 gttaatggga atgtattaca attcaagggt aacgatgaattaacgctatt aacttcttct 3300 agtacagtaa acgttgatgt aacagctgat ggaattacaaaacgtattcc agtaaaatat 3360 atcaactctg caagtgtacc tgccagtgca acagtagcaacaagtcctgt tactgttaag 3420 cttaattcaa gtgataatga tttaacattt gaagaattaatattcggtgt aattgaccct 3480 acacaattag tcaaagatga agacatcaac gaatttattgcagtttcaaa agcggctaaa 3540 aatgatggat atttgtataa taaaccgctt gtaacggttaaagatgcatc aggaaaagtt 3600 attccaacag gtgcaaatgt ttacggtcta aatcatgatgcaactaacgg aaacatttgg 3660 tttgatgagg aacaagctgg cttagctaaa aaatttagtgatgtacattt tgatgttgat 3720 ttttcattag ctaacgttgt aaaaactggt agcggtacagtttcttcatc gccatcatta 3780 tctgacgcaa ttcaacttac taattcaggc gatgcagtatcgtttacatt agttatcaaa 3840 tcaatttatg ttaaaggcgc agataaagat gataataacttacttgcagc ccctgtttct 3900 gtcaatgtga ctgtgacaaa ataattttga ggttcggtctctgttaccat ttgaaaaatg 3960 ccgaaaagct ctgcggagag aaatctctgc ggggcttttctttttggttc tatgtcaatt 4020 gttgaggtgc atggatcctc tagagtcgac ctgcaggcaagcttg 4065

What is claimed is:
 1. A process for producing S-layer proteins, whichcomprises (a) preparing a Gram-negative prokaryotic host cell which istransformed with a nucleic acid which codes for an S-layer protein froma non-Gram negative bacteria and is operatively linked to a signalsequence which codes for a peptide which brings about integration of theS-layer protein in the outer membrane of the host cell, integration ofthe S-layer protein in the cytoplasmic membrane of the host cell,secretion of the S-layer protein into the periplasmic space of the hostcell or/and secretion into the medium surrounding the host cell, (b)cultivating the host cell under conditions leading to expression of thenucleic acid and to production of the polypeptide encoded thereby, and(c) where appropriate isolating the resulting polypeptide from the outermembrane of the host cell, from the cytoplasmic membrane of the hostcell from the periplasmic space of the host cell or/and from the mediumsurrounding the host cell.
 2. A process as claimed in claim 1, whereinan Esherichia coli host cell is used.
 3. A process as claimed in claim1, wherein the nucleic acid which codes for an S-layer protein, codesfor a self assembling S-layer protein and is selected from (i) a nucleicacid encoding a SbsA protein wherein said nucleic acid comprises thenucleotide sequence shown in SEQ ID NO: 1 from position 91 to 3684, (ii)a nucleic acid which comprises a nucleotide sequence corresponding tothe nucleic acid from (i) within the scope of the degeneracy of thegenetic code, and (iii) a nucleic acid comprising a nucleotide sequencewhich hybridizes with a nucleic acid sequence which is complementary tothe nucleic acids from (i) and/or (ii) after washing at 55° C. in0.2×SSC buffer.
 4. A process as claimed in claim 1, wherein the nucleicacid coding for the S-layer protein comprises one or more insertionswhich code for heterologous peptide or polypeptide sequences.
 5. Aprocess as claimed in claim 4, wherein the insertion site is located atone or more positions selected from the group consisting of position562, 585, 881, 920, 1087, 1813, 1947, 2295, 2652, 3046, 3484, and 3594of the nucleotide sequence shown in SEQ ID NO:1.
 6. A process as claimedin claim 4, wherein the insertions are selected from nucleotidesequences which code for cysteine residues, regions with several chargedamino acids or Tyr residues, DNA-binding epitopes, metal-bindingepitopes, immunogenic epitopes, allergenic epitopes, antigenic epitopes,streptavidin, enzymes, cytokines or antibody-binding proteins.
 7. Aprocess as claimed in claim 6, wherein the insertions code forimmunogenic epitopes from herpes viruses, FMDV, flaviviruses orfiloviruses.
 8. A process as claimed in claim 1, wherein an operativelylinked nucleic acid which codes for a signal peptide from Gram-negativeprokaryotic cells is located on the 5′ side of the nucleic acid codingfor the S-layer protein.
 9. A process as claimed in claim 8, wherein thenucleic acid coding for the signal peptide comprises (a) the signalpeptide encoding section of the nucleotide sequence depicted in SEQ IDNO: 7 or 8, (b) a nucleotide sequence corresponding to the sequence from(a) within the scope of degeneracy of the genetic code or/and (c) anucleotide sequence which is at least 80% homologous with the sequencefrom (a) or (b).
 10. A process as claimed in claim 1, wherein at leasttwo S-layer genes are expressed in the host cell, one of which codes fora modified S-layer protein and another codes for an unmodified S-layerprotein.
 11. A process as claimed in claim 10, wherein the modifiedS-layer protein is able to form an S-layer structure which is compatiblewith the unmodified S-layer protein.
 12. The process according to claim6, wherein said herpes viruses is selected from the group consisting ofherpes viruses 1 and 6.